US20210341492A1

US20210341492A1 - Newborn screening for primary immunodeficiencies, cystinosis, and wilson disease

Info

Publication number: US20210341492A1
Application number: US17/282,989
Authority: US
Inventors: Sihoun Hahn; Sunhee Jung; Jeffrey Whiteaker; Troy Torgerson; Amanda Paulovich; Christopher Collins; Remwilyn Dayuha
Original assignee: Fred Hutchinson Cancer Research Center; Seattle Childrens Hospital
Current assignee: Fred Hutchinson Cancer Center
Priority date: 2018-10-05
Filing date: 2019-10-04
Publication date: 2021-11-04
Also published as: JP2022512591A; EP3861018A4; CN112805299A; EP3861018A1; WO2020072996A1

Abstract

Newborn screening for primary immunodeficiencies, cystinosis, and Wilson disease is described. The newborn screening can detect these disorders from dried blood spots already routinely collected at the time of birth. Early detection of these disorders will greatly improve patient outcome as each of them can be fatal once symptoms emerge.

Description

CROSS-REFERENCE TO RELATED APPLICATION

This application claims priority to U.S. Provisional Patent Application No. 62/742,161 filed Oct. 5, 2018, which is incorporated herein by reference in its entirety as if fully set forth herein.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with government support under Grant no. 1R21HD069890-01A1, Grant no. A1106784-01A1, and Grant no. R01A1123135, all awarded by the National Institutes of Health. The government has certain rights in the invention.

STATEMENT REGARDING SEQUENCE LISTING

The Sequence Listing associated with this application is provided in text format in lieu of a paper copy, and is hereby incorporated by reference into the specification. The name of the text file containing the Sequence Listing is 2698490_ST25.txt. The text file is 146 KB, was created on Oct. 1, 2019, and is being submitted electronically via EFS-Web.

FIELD OF THE DISCLOSURE

The current disclosure provides newborn screening for primary immunodeficiencies, cystinosis, and Wilson disease. The newborn screening can detect these disorders from dried blood spots already routinely collected at the time of birth. Early detection of these disorders will greatly improve patient outcome as each of them can be fatal once symptoms emerge.

BACKGROUND OF THE DISCLOSURE

There are a number of diseases with effective treatments available. However, for a number of these diseases, once symptoms emerge, the disease is already fatal or has led to irreversible damage. Examples of such diseases include primary immunodeficiency diseases (PIDD), cystinosis, and Wilson Disease (WD).
PIDD are a group of life-threatening hereditary or genetic congenital diseases characterized by an absent, impaired, or non-functioning immune system. Examples of PIDD include severe combined immunodeficiency (SCID), Wiskott-Aldrich Syndrome (WAS), and X-linked agammaglobulinemia (XLA).
SCID is a group of rare disorders caused by mutations in different genes involved in the development and function of infection-fighting immune cells such as T cells and B cells. SCID patients are usually affected by severe bacterial, viral, or fungal infections early in life and often are afflicted with scarring of the lungs, chronic diarrhea, and failure to thrive. The condition is fatal, usually within the first year or two of life, unless infants receive immune-restoring treatments, such as transplants of blood-forming stem cells, gene therapy, or enzyme therapy.
WAS is an immune deficiency that primarily affect males and is characterized by a reduced ability to form blood clots, due to a decrease in the number and size of platelets. This platelet abnormality, which is typically present from birth, leads to easy bruising and episodes of prolonged bleeding following minor trauma, which in some cases, is life threatening. Individuals with WAS also have an increased risk of developing infections, autoimmune disorders, and certain types of cancer. In WAS patients, immunoglobulin infusions and antibiotics can be used to prevent infections, and in severe cases, stem cell transplantation can provide a cure. Gene therapies are also being explored as another alternative treatment.
XLA is an inherited immunodeficiency that also primarily affect males. In XLA, the body is unable to produce antibodies needed to defend against bacteria, viruses, and other foreign substances. Children with XLA are usually healthy for the first 1 or 2 months of life because they are protected by maternal antibodies acquired before birth. After this time, however, the maternal antibodies are cleared from the body, and the affected child develops recurrent infections that lead to organ damage. Once the disease is detected, immunoglobulin infusions provide a standard therapy to strengthen the immune system, and antibiotics can be given to fight bacterial infections. However, by the time a diagnosis is made, organ damage may have already occurred.
Cystinosis is a rare metabolic disorder in which the amino acid cystine gets into cells but cannot exit due to defects in a cystine-specific transporter called cystinosin. Because of the defect in transportation of cystine, cells accumulate cystine in crystals in lysosomes, organelles inside cells, leading to early cell death. Cystinosis slowly destroys the organs in the body including the kidneys, liver, eyes, muscles and the brain. Children with infantile cystinosis appear normal at birth, but by 9-10 months of age, have symptoms that include excessive thirst and urination and failure to thrive. Cysteamine is a treatment that slows the progression of cystinosis by removing cystine from the cells. If cysteamine treatment is not started early enough in disease progression, however, renal transplantation is usually required.
WD is a copper transport disorder in which copper accumulates in vital organs such as the liver, kidneys, and brain. Diagnosing WD is difficult because its progression is slow and there is a broad spectrum of clinical symptoms. Therefore, despite the fact that treatments are available, many patients still present with irreversible multi-organ damage at the time of diagnosis.
The treatment of each of the above-described diseases would be significantly enhanced if diagnosis could be made before clinical symptoms emerge. Newborn screening (NBS) is a standard public preventive mandatory screening test for the 4 million babies born every year in the U.S. NBS usually involves a blood test performed 24 to 48 hours after birth. The screening uses a few drops of blood from a newborn's heel deposited on filter paper. The paper containing dried blood spots (DBS) can be stored until the tests are conducted.
To conduct NBS assessments, punches of dried blood are taken from the DBS and laboratory tests are performed to detect the presence or absence of specific substances within the blood (called markers or biomarkers) that are indicative of disorders not apparent at birth but that cause serious health problems later in life. Though the disorders screened vary from state to state, most states screen for phenylketonuria, primary congenital hypothyroidism, cystic fibrosis, and sickle cell disease. NBS has proven to be highly effective at improving patient outcomes and avoiding long-term disability in affected individuals, while at the same time reducing healthcare costs.
SCID has recently been added to NBS panels. However, the current molecular test for SCID is not able to reliably identify all patients having SCID-like disorders including late and delayed onset adenosine deaminase (ADA) deficiency, ZAP70 deficiency, and MHC Class II deficiency. Unfortunately, for most of the other life-threatening but treatable “non-SCID” immunodeficiencies, there are currently no broad-based, cost-effective screening methods available.
One reason that many disorders that would be beneficial to screen for lack an available test is that laboratory tests have been unable to reliably measure markers associated with the disorders. One reason for this inability is that the disorders are associated with the absence of or very reduced levels of markers. In this scenario, it can be difficult to reliably detect the absence of markers due to “background noise” associated with many laboratory techniques that can mask clinically relevant results.
Peptide immunoaffinity enrichment coupled to selected reaction monitoring mass spectrometry (immuno-SRM) is a method that enables precise quantification of low abundance markers. Utilization of immuno-SRM generally involves the following steps: (i) selection of markers that are indicative of the presence or absence of a disorder; (ii) treatment of a biological sample that would include the marker, if present, with enzymes to digest all proteins in the biological sample into smaller fragments called peptides; (iii) enrichment for peptides derived from the selected marker and (iv) analysis and quantification of the enriched peptides of interest in a mass spectrometer.
Recently, Jung et al., (J. Proteome Res. 2017; 16: 862-871) described use of immuno-SRM to quantify a protein marker, ATP7B, associated with WD from DBS. This was the first demonstration that immuno-SRM could be used to reliably detect a low-abundant marker from stored DBS, opening the possibility of using the approach to screen for a wider panel of disorders.
Though Jung and colleagues demonstrated the feasibility of using DBS in an immuno-SRM assay to distinguish individuals affected by WD from unaffected individuals, many aspects of such an assay depend on the disorder being diagnosed, the biomarkers available for each disorder, the ability to develop molecular entities that can enrich for peptides of interest, and the behavior of each peptide of interest in the mass spectrometer. All of these aspects and more require careful consideration and experimentation to achieve an assay that can reliably detect disorders in an NBS panel using DBS before clinical symptoms emerge.

SUMMARY OF THE DISCLOSURE

The current disclosure describes development of multiplexed assays that can be used to screen newborns for severe combined immunodeficiency (SCID), Wiskott-Aldrich Syndrome (WAS), X-linked agammaglobulinemia (XLA), cystinosis, and Wilson Disease (WD). The assays can significantly improve the outcome for affected individuals by reliably diagnosing these disorders before devastating and often fatal clinical symptoms emerge. The assays can detect the presence or absence of markers associated with these disorders using dried blood spots (DBS) already routinely collected as part of existing newborn screening (NBS) procedures.
The current disclosure describes peptides associated with each of the disorders that can be reliably detected and quantified using peptide immunoaffinity enrichment coupled to selected reaction monitoring mass spectrometry (immuno-SRM). The current disclosure also provides high affinity antibodies that can be used to enrich for the peptides, as well as methods to tag the peptides to increase the throughput of the assay.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

FIG. 1. Protein targets and peptide sequences used for peptide immunoaffinity enrichment coupled to selected reaction monitoring mass spectrometry (immuno-SRM-MS) to diagnose severe combined immunodeficiency (SCID), Wiskott-Aldrich Syndrome (WAS), X-linked agammaglobulinemia (XLA), cystinosis, and Wilson Disease (WD). Total mass, parent ion mass, and daughter ion masses are also shown. ++ indicates doubly charged parent ions. The ion type for daughter ions are in parenthesis. *Total mass and parent ion mass when detecting ragged end sequence with leading K amino acid residue.

FIGS. 2A-2E. Response curves for peptides measured by the multiplexed immuno-multiple reaction monitoring (MRM) assay. When multiple parent ions are monitored in a single mass spectrometry (MS) run, this type of analysis is known as multiple reaction monitoring (MRM). Using MRM analysis, multiple proteins and multiple regions (signature peptides) of a protein can be monitored in a single mass spectrometry run. Response curves plot the heavy:light peak area ratio as a function of heavy peptide concentration, measured in a background matrix of digested protein extracted from dried blood spots (DBS). The curves allow determination of the linear range and sensitivity of the assay. Each datapoint is plotted as a gray box and linear regression is plotted as a line. Regression fit parameters are reported in the corner of each plot. Weighting for each plot is 1/x. (FIG. 2A) BTK 407; (FIG. 2B) BTK 545; (FIG. 2C) CD3ε 197; (FIG. 2D) WASp 274; (FIG. 2E) WASp 289.

FIGS. 3A-3E. MRM traces for internal standard (left panels) and endogenous (right panels) signature peptides: (FIG. 3A) CD3ε 197; (FIG. 3B) BTK 407; (FIG. 3C) BTK 545; (FIG. 3D) WASp 274; (FIG. 3E) WASp 289.

FIGS. 4A-4D. Inter-laboratory correlation in measured PIDD peptide concentrations. (FIG. 4A) WASp 274; (FIG. 4B) CD3ε 197; (FIG. 4C) BTK 407; (FIG. 4D) ATP7B 1056.

FIG. 5. Inter-laboratory analytical validation of WASp 289.

FIGS. 6A-6E. Differences in signature peptide levels between patients (Pt). (FIG. 6A) BTK 545; (FIG. 6B) BTK 407; (FIG. 6C) CD3ε 197; (FIG. 6D) WASp 274; (FIG. 6E) WASp 289 (SCID: n=3, WAS: n=11, BTK: n=26) and normal controls (NC, n=40). **** p<0.0001, *p<0.05.

FIG. 7. ATP7B 1056 signature peptide concentrations.

FIG. 8. Quantification of signature peptides in normal controls from a blinded cohort study.

FIG. 9. Concentrations of signature peptides in a blinded patient cohort study.

FIGS. 10A, 10B. Receiver operating characteristic (ROC) plots showing the diagnostic performance of immuno-SRM for PIDDs. (FIG. 10A) ROC plots for BTK 407, BTK 545, CD3ε 197, WASp 274, and WASp 289. True positive and false positive rates are plotted for increasingly stringent cutoff values. Line of identity indicates a test that cannot distinguish patients from controls. (FIG. 10B) Area under the curve (AUC) values and p-values for peptides shown in FIG. 10A.

FIG. 11. Signature peptide levels in DBS obtained from Washington State Newborn Screening Laboratory.

FIG. 12. Ratios of signature peptides against ATP7B peptide and patient diagnosis in a blinded cohort study.

FIG. 13. Concentrations of CTNS and SHPK proteins in patients and normal control samples. Samples outlined in black have confirmed mutational status while samples outlined in gray are predicted to be homozygous for the 57-kb deletion. Samples CTNS 00001-CTNS 00013 are blinded and predicted genotype is based on protein levels evidenced by immuno-SRM.

FIG. 14. Comparison of the signal-to-noise (S/N) ratios achieved using both micro-flow ionkey detection methods and new nano-flow methods for CTNS 115. Nano-liter per minute flow rates boost the signal produced by CTNS 115 target peptide.

FIG. 15. Measured CTNS 115 and SHPK 363 concentrations in DBS and predicted genotype by immuno-SRM. Peptides were considered not detected (N.D.) if the signal-to-noise ratio for the peptide was less than 10. * indicates unknown mutations at this time.

FIGS. 16A-16D. Internal standard peptide peak areas generated upon peptide capture of both WASp 274 (FIGS. 16B and 16D) and BTK 407 (FIGS. 16A and 16C) using polyclonal and monoclonal antibodies. Peak areas were measured in blank control samples only (i.e. no patient blood, FIGS. 16C, 16D) and in an aggregated set of both patient and blank control samples (FIGS. 16A and 16B).

FIG. 17. Signature CTNS 115 and SHPK 363 peptide levels in patients and normal control (NC) samples in a screen for cystinosis. The study is an extension of that shown in FIG. 13 and Table 8.

FIGS. 18A-18C. A study of 16 WD patients (NC: normal control; Pt: Wilson disease patients). (FIG. 18A) ATP7B 1056 concentration in NC and WD patients. The 50 NC in the first column were run separately to set the reference range in the lab; the 3 NC in the middle column were run together with all 16 WD patients as quality control. (FIG. 18B) ATP7B 214 concentration in NC and WD patients. (FIG. 18C) ATP7B 887 concentration in NC and WD patients.

FIG. 19. Sequences (SEQ ID NOs: 22-76, and 78-141) supporting the disclosure.

DETAILED DESCRIPTION

There are a number of diseases with effective treatments available. However, for a number of these diseases, once symptoms emerge, the disease is already fatal or has led to irreversible damage. Examples of such diseases include primary immunodeficiency diseases (PIDD), cystinosis, and Wilson Disease (WD).
PIDD are a collection of diverse congenital diseases characterized by absent or impaired immune responses including: autosomal recessive CD3ε-associated severe combined immunodeficiency (SCID, OMIM #615615); X-linked Wiskott-Aldrich Syndrome (WAS, OMIM #301000); and X-linked agammaglobulinemia (XLA, OMIM #300755). Although genetically and clinically heterogeneous, these disorders lead to fatal infections unless detected and treated early. While early detection of PIDD can be life-saving, unfortunately, most affected infants are diagnosed only after developing devastating infections.
SCID is a group of rare disorders caused by mutations in different genes involved in the development and function of infection-fighting immune cells such as T cells and B cells. More than a dozen genes have been implicated in SCID. Most often SCID is inherited in an autosomal recessive pattern, in which both copies of a particular gene, one inherited from the mother and one from the father, contain defects. The best-known form of autosomal recessive SCID is caused by adenosine deaminase (ADA) deficiency, in which infants lack the ADA enzyme necessary for T-cell survival. X-linked SCID, which is caused by mutations in a gene on the X chromosome, primarily affects males. Boys with this type of SCID have white blood cells that grow and develop abnormally. As a consequence, they have low numbers of T cells and natural killer cells, and their B cells do not function. SCID patients are usually affected by severe bacterial, viral, or fungal infections early in life and often are afflicted with scarring of the lungs, chronic diarrhea, and failure to thrive. The condition is fatal, usually within the first year or two of life, unless infants receive immune-restoring treatments, such as transplants of blood-forming stem cells, gene therapy, or enzyme therapy.
WAS is an immune deficiency that is characterized by a decrease in the number and size of platelets. WAS is caused by mutations in the WAS gene which produces the WAS protein (WASp), and is often considered to be part of a disease spectrum with two other disorders: X-linked thrombocytopenia and severe congenital neutropenia. These conditions have overlapping signs and symptoms and the same genetic cause.
The decrease in the number and size of platelets associated with WAS results in a reduced ability to form blood clots. This leads to easy bruising and episodes of prolonged bleeding following minor trauma, which in some cases, is life threatening. Individuals with WAS also have an increased susceptibility to infection, autoimmune disorders (e.g., eczema) and certain cancers (e.g., lymphoma). Once diagnosed, treatments for WAS are available. Exemplary treatments include immunoglobulin infusions, antibiotics, and stem cell transplants. Gene therapy is also being explored as a treatment option for WAS.
XLA is an inherited immunodeficiency which prevents B cells from developing normally. XLA is caused by mutations in a gene called Bruton's Tyrosine Kinase (BTK). XLA results in an inability to produce antibodies needed to defend against bacteria, viruses, and other foreign substances. Children with XLA are usually healthy for the first 1 or 2 months of life because they are protected by maternal antibodies acquired before birth. After this time, however, the maternal antibodies are cleared from the body, and the affected child develops recurrent infections. Recurrent infections can lead to organ damage. Once diagnosed, treatments in the form of antibody infusions and antibiotics are available to treat XLA.
Cystinosis is a rare metabolic disorder in which the amino acid cystine gets into cells but cannot exit due to defects in a cystine-specific transporter called cystinosin. Because of the defect in transportation of cystine, cells accumulate cystine in crystals in lysosomes, organelles inside cells, leading to early cell death. Over 90 mutations in the cystinosin (CTNS) gene or region have been reported. About half of the cystinosis variant genes in the Western populations are caused by a large chromosomal deletion which extends from part of the CTNS gene through the adjacent SHPK and initial part of the TRPV1 (capsaicin receptor) genes. Cystinosis slowly destroys the organs in the body including the kidneys, liver, eyes, muscles and the brain. Children with infantile cystinosis appear normal at birth, but by 9-10 months of age, have symptoms that include excessive thirst and urination and failure to thrive. If diagnosed early, treatment with cysteamine, a cystine depleting agent that slows the progression of cystinosis by removing cystine from the cells, is possible. If not diagnosed early enough, renal transplantation is often required.
Wilson Disease (WD) is a slow and progressive copper transport disorder in which excess copper is not eliminated properly and accumulates in vital organs such as the liver, kidneys, and brain. WD is caused by a genetic defect in the ATP7B gene. WD leads to irreversible neurological disability and liver cirrhosis if not diagnosed and treated early. Unfortunately, WD remains difficult because of its slow progression and the broad clinical spectrum of symptoms. Therefore, many patients still present with irreversible and sometimes fatal multi-organ damage at the time of diagnosis. When diagnosed early enough, available treatments include D-penicillamine, trientine and zinc salts.
The treatment of each of the above-described diseases would be significantly enhanced if diagnosis could be made before clinical symptoms emerge. Newborn screening (NBS) is a standard public preventive mandatory screening test for the 4 million babies born every year in the U.S. NBS usually involves a blood test performed 24 to 48 hours after birth. The screening uses a few drops of blood from a newborn's heel deposited on filter paper. The paper containing dried blood spots (DBS) can be stored until the tests are conducted.
To conduct NBS assessments, punches of dried blood are taken from the DBS and laboratory tests are performed to detect the presence or absence of specific substances within the blood (called markers or biomarkers) that are indicative of disorders not apparent at birth but that cause serious health problems later in life. Though the disorders screened vary from state to state, most states screen for phenylketonuria, primary congenital hypothyroidism, cystic fibrosis, and sickle cell disease. NBS has proven to be highly effective at improving patient outcomes and avoiding long-term disability in affected individuals, while at the same time reducing healthcare costs.
T-cell receptor excision circle (TREC) analysis and kappa-deleting element recombination circle (KREC) screening from dried blood spots (DBS) on filter paper has recently been introduced for SCID and some X-linked or autosomal recessive agammaglobulinemias. However, NBS methods for other PIDD do not exist.
Tandem mass spectrometry (MS/MS) was first applied to NBS in the 1990s, paving the way for rapid screening of multiple metabolites and thus several diseases from DBS samples collected at birth. Chace J Mass Spectrom. Wiley-Blackwell; 2009; 44: 163-170; Millington et al. J. Inherit. Metab. Dis. 1990; 13: 321-324; Sweetman et al. Pediatrics. 2006; 117: S308-S314; Almannai et al. Curr. Opin. Pediatr. 2016; 28: 694-699; Watson et al. Genet. Med. Nature Publishing Group; 2006. pp. 1S-252S; Chace et al. Clin. Chem. 1993; 39: 66-71. Selected reaction monitoring mass spectrometry (SRM-MS) performed on triple quadrupole mass spectrometers further enabled the precise, high-throughput, and analytically-robust quantification of specific biomarkers; as such, it is now the standard of care at clinical NBS laboratories across the world. Chace D H J Mass Spectrom. Wiley-Blackwell; 2009; 44: 163-170; Chace & Kalas. Clinical Biochemistry. 2005; 38: 296-309. Dott et al. American Journal of Medical Genetics Part A. Wiley Subscription Services, Inc., A Wiley Company; 2006; 140: 837-842.
MS/MS relies on the measurement of concentrated upstream metabolites for detection of various inborn errors of metabolism with specific enzyme deficiencies. This excludes its application to diseases, such as PIDD, where no accumulated metabolites are present or currently verified. For this reason, protein-based assays such as flow cytometry or western blotting have been used as first-line investigative methods for diseases such as WAS and its milder phenotype, X-linked thrombocytopenia (XLT), where most mutations lead to absent or decreased protein products. Qasim et al. Br. J. Haematol. 2001; 113: 861-865; Jin et al. Blood. American Society of Hematology; 2004; 104: 4010-4019. These approaches require that intact blood samples or cells from patients be available, making population-based screening or testing of patients from resource-poor areas impossible.
SRM-MS utilizes proteolytically-generated signature peptides as stoichiometric surrogates of a protein of interest. This may, in turn, be used to estimate the number of a particular cell-type expressing that protein in a sample (i.e. quantification of CD3ε for an indication of the amount of CD3+ T-cells in blood). The high specificity of MS for each signature peptide is conferred by three physiochemical properties—its mass, retention times upon high-performance liquid chromatography (HPLC) separation, and resultant target-specific fragmentation patterns. Kennedy et al. Nat. Methods. 2014; 11: 149-155. Despite these advances, with a typical limit of quantification ranging from 100 to 1000 ng protein/mL, the use of complex matrices such as blood or plasma often precludes accurate quantification of low-abundance targets by SRM-MS based assays. This limits applicability to many PIDD including SCID, WAS, and XLA that result in absent or decreased levels of target proteins expressed only intracellularly. de Saint Basile et al. J. Clin. Invest. American Society for Clinical Investigation; 2004; 114: 1512-1517.
Peptide immunoaffinity enrichment coupled to SRM (immuno-SRM), also referred to as Stable Isotope Standards and Capture by Anti-Peptide Antibodies (SISCAPA), increases the sensitivity of SRM-MS assays by utilizing anti-peptide antibodies to purify and enrich peptides of interest from a complex biologic sample prior to SRM-MS analysis. Zhao et al. J Vis Exp. 2011; 53: 2812; Whiteaker et al. Mol. Cell Proteomics. American Society for Biochemistry and Molecular Biology; 2010; 9: 184-196; Whiteaker et al. Mol. Cell Proteomics. American Society for Biochemistry and Molecular Biology; 2012; 11: M111.015347; Kuhn et al. Clin. Chem. 2009; 55: 1108-1117; Anderson et al. J Proteome Res. 2004; 3(2): 235-244. This additional peptide enrichment step, coupled to SRM-MS, lowers the limit of detection to the low pg protein/mL range from 1 mL of plasma that is suitable for the accurate quantification of very low abundance proteins in complex matrices such as DBS. Whiteaker et al. Mol. Cell Proteomics. American Society for Biochemistry and Molecular Biology; 2010; 9: 184-196; Hoofnagle et al. Clin. Chem. 2008; 54: 1796-1804; Netzel et al. Clin. Chem. 2016; 62: 297-299; Razavi et al. Bioanalysis. Future Science Ltd London, UK; 2016; 8: 1597-1609.
The ability of LC-MS/MS to detect signature peptides from CD3ε, WASp, and BTK in proteolytically digested human peripheral blood mononuclear cell (PMBC) lysates has previously been demonstrated. In a blinded study, peptide levels were quantified in normal control PMBCs but nearly undetectable in a disease-specific fashion in affected patients. Kerfoot et al. Proteomics Clin Appl. 2012; 6: 394-402. The same proteomic method was applied to show elevated levels of α-aminoadipic semialdehyde antiquitin (α-AASA) and piperideine-6-carboxylate (P6C) in DBS of patients with pyridoxine-dependent seizures, revealing the possibility of its application to NBS. Jung et al. Mol. Genet. Metab. 2013; 110: 237-240. To improve the sensitivity and reproducibility of the assay, the immuno-SRM platform was harnessed to allow for the quantification of very low abundance peptides in DBS such as surrogate peptides of the ATP7B protein from patients with Wilson Disease (WD). The results demonstrated the ability of immuno-SRM to detect ATP7B peptides in the low picomolar (pmol) range and to reproducibly differentiate between patients with WD from unaffected individuals. Jung et al. 2017, supra.
Though the feasibility of using DBS in an immuno-SRM assay to distinguish individuals affected by certain diseases from unaffected individuals has been established, many aspects of the assay depend on the disorder being diagnosed, the biomarkers available for each disorder, the ability to develop molecular entities that can enrich for peptides of interest, and the behavior of each peptide of interest in the mass spectrometer. All of these aspects and more require careful consideration and experimentation to achieve a reliable assay that can reliably detect disorders in an NBS panel using DBS before clinical symptoms emerge.
The present disclosure provides a multiplexed immuno-SRM method to reliably diagnose SCID, WAS, XLA, cystinosis, and WD from a dried blood spot (DBS). The multiplexed immuno-SRM assay disclosed herein can utilize anti-peptide antibodies generated against peptides of proteins reduced or absent in SCID, WAS, XLA, cystinosis, and WD.
The following aspects of the disclosure are now described in more detail: (I) Collection and Processing of DBS; (II) Peptide Markers for SCID, WAS, XLA, cystinosis, and Wilson disease; (III) Enzymatic Digestion of Proteins in DBS; (IV) Antibodies to Enrich for the Peptide Markers; (V) Enrichment Strategies for Peptides; (VI) Liquid Chromatography (LC); (VII) Mass Spectrometry (MS); (VIII) Kits; (IX) Methods of Use; (X) Exemplary Embodiments; and (XI) Experimental Examples.

(I) COLLECTION AND PROCESSING OF DBS

In particular embodiments, samples used in the methods of the present disclosure are DBS. In particular embodiments, whole blood from a subject can be prepared by placing blood onto a filter paper card and allowing the blood to dry.
In particular embodiments, whole blood from a subject can be collected in ACD (acid citrate dextrose) tubes and DBS can be prepared by pipetting 50-100 μL (e.g., 70 μL) blood/spot onto filter paper card (e.g., Protein Saver™ 903® Card, Whatman Inc, Piscataway, N.J.), and allowed to dry at room temperature. In particular embodiments, blood is allowed to dry on filter paper card overnight. DBS can be stored, for example, in sealed plastic bags at −80° C. until use. In particular embodiments, the whole DBS can be used in the immuno-SRM assays of the disclosure. In particular embodiments, one or more 3-mm punches from the DBS can be used in the immuno-SRM assays of the disclosure.

(II) PEPTIDE MARKERS FOR SCID, WAS, XLA, CYSTINOSIS, AND WD

There are many theoretical proteolytic peptides from target proteins. Those can be potential candidates for monoclonal antibody production. Nonetheless, the best potential candidate peptides were chosen after screening their characteristics by MS/MS. Those signature peptides with the highest sensitivity and specificity were selected to develop corresponding monoclonal antibodies and validated using clinical samples. In particular embodiments, multiple peptides and antibodies can be included in a multiplex analysis to maximize the sensitivity and specificity of the SRM assay.
Typically, one or two signature proteotypic peptides which are unique to the protein of interest and that are consistently observed in MS experiments are selected to stoichiometrically represent the protein of interest. Mallick et al. Nat Biotechnol 2007; 25: 125-131. Signature peptides can be selected by detection in previous MS experiments, use of computational tools to predict the peptides most likely observable by MS, or a combination of both. In particular embodiments, tryptic peptides 8-22 amino acids in length with moderate hydrophobicity can be selected. Very hydrophilic and very hydrophobic peptides can be less stable due to retention time variation in HPLC and loss to surfaces. In particular embodiments, Methionine residues (oxidation), N-terminal glutamine (cyclization), asparagine followed by glycine or proline (prone to deamidation), and dibasic termini (e.g. neighboring lysine or arginine residues such as KK, KR, RR, RK have the potential for variable digestion efficiency) can be undesirable. Whiteaker and Paulovich Clin Lab Med. 2011; 31(3): 385-396. Shorter peptides and those containing proline residues can be better targets for SRM. Lange et al. Molecular Systems Biology 2008; 4: 222.
In particular embodiments, the peptides include portions of CD3ε, WASp, BTK, CTNS, SHPK and/or ATP7B. In particular embodiments, the peptides include SEQ ID NOs: 1-21.
In particular embodiments, peptides of the present disclosure include: CD3ε 197 for SCID; WASp 274 for WAS; WASp 289 for WAS; BTK 407 for XLA; BTK 545 for XLA; CTNS 115 for cystinosis; CTNS 120 for cystinosis; CTNS 194 for cystinosis; CTNS 360 for cystinosis; SHPK 44 for cystinosis; SHPK 363 for cystinosis; SHPK 388 for cystinosis; ATP7B 214 for WD; ATP7B 325 for WD; ATP7B 466 for WD; ATP7B 589 for WD; ATP7B 621 for WD; ATP7B 887 for WD; ATP7B 1056 for WD; and ATP7B 1061 for WD. In particular embodiments, proteolytically-generated signature peptides useful for methods of the present disclosure include peptides in FIG. 1: CD3ε 197 (DLYSGLNQR, SEQ ID NO: 1); WASp 274 (AGISEAQLTDAETSK, SEQ ID NO: 2); WASp 289 (LIYDFIEDQGGLEAVR, SEQ ID NO: 3); BTK 407 (ELGTGQFGVVK, SEQ ID NO: 4); BTK 545 (YVLDDEYTSSVGSK, SEQ ID NO: 5); CTNS 115 (FLVIR, SEQ ID NO: 6); CTNS 360 (RPGYDQLN (SEQ ID NO: 7) and KRPGYDQLN (SEQ ID NO: 8)); SHPK 363 (DTHLTITPTVLGER, SEQ ID NO: 9); ATP7B 325 (VSLPDGAEGSGTDHR, SEQ ID NO: 10); and ATP7B 1056 (VLAVVGTAEASSEHPLGVAVTK, SEQ ID NO: 11). In particular embodiments, additional peptides useful for methods of the present disclosure include the following in Table 1.

TABLE 1

Additional exemplary peptides of the
present disclosure

Peptide name	Sequence

CTNS 120-151	SSAISIINQVIGWIYFVAWSISFYPQVIMNWR
“CTNS 120”	(SEQ ID NO: 12)

CTNS 194-228	YPNGVNPVNSNDVFFSLHAVVLTLIIIVQC(CAM)
“CTNS 194”	C(CAM)LYER (SEQID NO: 13)

SHPK 44-58	AEAAVESAVAGPQGR (SEQ ID NO: 14)
“SHPK 44”

SHPK 388-400	ISSSDLSLGHVTR (SEQ ID NO: 15)
“SHPK 388”

ATP7B 214-226	VAPLSLGPIDIER (SEQ ID NO: 16)
“ATP7B 214”

ATP7B 466-477	LPANHAPDILAK (SEQ ID NO: 17)
“ATP7B 466”

ATP7B 589-603	TNGITYASVALATSK (SEQ ID NO: 18)
“ATP7B 589”

ATP7B 621-634	IIEEIGFHASLAQR (SEQ ID NO: 19)
“ATP7B	621”

ATP7B 887-901	ATHVGNDTTLAQIVK (SEQ ID NO: 20)
“ATP7B 887”

ATP7B 1061-1077	GTAEASSEHPLGVAVTK (SEQ ID NO: 21)
“ATP7B 1061”

(III) ENZYMATIC DIGESTION OF PROTEINS IN DBS

Proteins in DBS can be subjected to proteolysis to produce peptides that can be further selected by immunoaffinity purification before analysis by LC-SRM-MS. Proteolysis can be accomplished using site specific endoproteases, such as pepsin, arg-C proteinase, asp-N endopeptidase, BNPS-skatole, caspase 1, caspase 2, caspase 3, caspase 4, caspase 5, caspase 6, caspase 7, caspase 8, caspase 9, caspase 10, chymotrypsin, clostripain (clostridiopeptidase B), enterokinase, factor Xa, glutamyl endopeptidase, granzyme B, lysC, proline-endopeptidase, proteinase K, staphylococcal peptidase I, thermolysin, thrombin, and trypsin. Chemicals which cleave site specifically can also be used. Combinations of enzymes and/or chemicals can be used to obtain desirable analytes.
In particular embodiments, proteins in DBS can be digested into peptides with trypsin. Trypsin cleaves exclusively C-terminal to arginine and lysine residues and can be a preferred choice to generate peptides because the masses of generated peptides are compatible with the detection ability of most mass spectrometers (up to 2000 m/z) and because there are efficient algorithms available for the generation of databases of theoretical trypsin-generated peptides. High cleavage specificity, availability, and cost are other advantages of trypsin. Peptides formed by the treatment of a protein with trypsin are known as tryptic peptides.

(IV) ANTIBODIES TO ENRICH FOR THE PEPTIDE MARKERS

An antibody includes a polypeptide ligand substantially encoded by an immunoglobulin gene or immunoglobulin genes, or fragments thereof, whether natural, or partially or wholly synthetically produced. An antibody specifically (or selectively) binds and recognizes an epitope (e.g., an antigen). An antibody can include any protein having a binding domain that is homologous or largely homologous to an immunoglobulin binding domain. An antibody may be monoclonal or polyclonal. The antibody may be a member of any immunoglobulin class, including any of the human classes: IgG, IgM, IgA, IgD, and IgE, etc. The recognized immunoglobulin genes include the kappa and lambda light chain constant region genes, the alpha, gamma, delta, epsilon and mu heavy chain constant region genes, and the myriad immunoglobulin variable region genes. “Fc” portion of an antibody refers to that portion of an immunoglobulin heavy chain that includes one or more heavy chain constant region domains, CH1, CH2 and CH3, but does not include the heavy chain variable region.
An intact antibody can include at least two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds. Each heavy chain is composed of a heavy chain variable region (abbreviated herein as VH or VH) and a heavy chain constant region. The heavy chain constant region includes three domains, CH1, CH2 and CH3. Each light chain is composed of a light chain variable region (abbreviated herein as VL or VL) and a light chain constant region. The light chain constant region includes one domain, CL. The VH and VL regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDR), interspersed with regions that are more conserved, termed framework regions (FR). Each VH and VL is composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain a binding domain that interacts with an antigen. The constant regions of the antibodies can mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (Clq) of the classical complement system.
The precise amino acid sequence boundaries of a given CDR or FR can be readily determined using any of a number of well-known schemes, including those described by: Kabat et al. (1991) “Sequences of Proteins of Immunological Interest,” 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (Kabat numbering scheme); Al-Lazikani et al. (1997) J Mol Biol 273: 927-948 (Chothia numbering scheme); Maccallum et al. (1996) J Mol Biol 262: 732-745 (Contact numbering scheme); Martin et al. (1989) Proc. Natl. Acad. Sci., 86: 9268-9272 (AbM numbering scheme); Lefranc M P et al. (2003) Dev Comp Immunol 27(1): 55-77 (IMGT numbering scheme); and Honegger and Pluckthun (2001) J Mol Biol 309(3): 657-670 (“Aho” numbering scheme). The boundaries of a given CDR or FR may vary depending on the scheme used for identification. For example, the Kabat scheme is based on structural alignments, while the Chothia scheme is based on structural information. Numbering for both the Kabat and Chothia schemes is based upon the most common antibody region sequence lengths, with insertions accommodated by insertion letters, for example, “30a,” and deletions appearing in some antibodies. The two schemes place certain insertions and deletions (“indels”) at different positions, resulting in differential numbering. The Contact scheme is based on analysis of complex crystal structures and is similar in many respects to the Chothia numbering scheme. In particular embodiments, the antibody CDR sequences disclosed herein are according to Kabat numbering.
An antibody fragment includes any derivative or portion of an antibody that is less than full-length. In particular embodiments, the antibody fragment retains at least a significant portion of the full-length antibody's specific binding ability as a binding partner. Examples of antibody fragments include Fab, Fab′, Fab′-SH, F(ab′)₂, single chain variable fragment (scFv), Fv, dsFv diabody, and Fd fragments, and/or any biologically effective fragments of an immunoglobulin that bind specifically to an epitope described herein. Antibodies or antibody fragments include all or a portion of polyclonal antibodies, monoclonal antibodies, human antibodies, humanized antibodies, synthetic antibodies, chimeric antibodies, bispecific antibodies, mini bodies, and linear antibodies.
A single chain variable fragment (scFv) is a fusion protein of the variable regions of the heavy and light chains of immunoglobulins connected with a short linker peptide. Fv fragments include the VL and VH domains of a single arm of an antibody. Although the two domains of the Fv fragment, VL and VH, are coded by separate genes, they can be joined, using, for example, recombinant methods, by a synthetic linker that enables them to be made as a single protein chain in which the VL and VH regions pair to form monovalent molecules (single chain Fv (scFv)). For additional information regarding Fv and scFv, see e.g., Bird, et al., Science 242 (1988) 423-426; Huston, et al., Proc. Natl. Acad. Sci. USA 85 (1988) 5879-5883; Plueckthun, in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore (eds.), Springer-Verlag, New York), (1994) 269-315; WO1993/16185; U.S. Pat. Nos. 5,571,894; and 5,587,458.
A Fab fragment is a monovalent antibody fragment including VL, VH, CL and CH1 domains. A F(ab′)₂fragment is a bivalent fragment including two Fab fragments linked by a disulfide bridge at the hinge region. For discussion of Fab and F(ab′)₂fragments having increased in vivo half-life, see U.S. Pat. No. 5,869,046. Diabodies include two epitope-binding sites that may be bivalent. See, for example, EP 0404097; WO1993/01161; and Holliger, et al., Proc. Natl. Acad. Sci. USA 90 (1993) 6444-6448. Dual affinity retargeting antibodies (DART™; based on the diabody format but featuring a C-terminal disulfide bridge for additional stabilization (Moore et al., Blood 117, 4542-51 (2011)) can also be used. Antibody fragments can also include isolated CDRs. For a review of antibody fragments, see Hudson, et al., Nat. Med. 9 (2003) 129-134.
The antibody fragment may be produced by any means. For example, the antibody fragment may be enzymatically or chemically produced by fragmentation of an intact antibody or it may be recombinantly produced from a gene encoding the partial antibody sequence. Alternatively, the antibody fragment may be wholly or partially synthetically produced. The antibody fragment may include a single chain antibody fragment. In another embodiment, the fragment may include multiple chains that are linked together, for example, by disulfide linkages. The fragment may also include a multimolecular complex. A functional antibody fragment may typically include at least 50 amino acids and more typically will include at least 200 amino acids.
In particular embodiments, recombinant immunoglobulins can be produced. See, Cabilly, U.S. Pat. No. 4,816,567, and Queen et al., Proc Natl Acad Sci USA, 86:10029-10033 (1989).
As indicated, in particular embodiments, binding domains of an engineered antibody or antigen binding fragment may be joined through a linker. A linker is an amino acid sequence which can provide flexibility and room for conformational movement between the binding domains of an engineered antibody or antigen binding fragment. Any appropriate linker may be used. Examples of linkers can be found in Chen et al., Adv Drug Deliv Rev. 2013 Oct. 15; 65(10): 1357-1369. Linkers can be flexible, rigid, or semi-rigid, depending on the desired functional domain presentation to a target. Commonly used flexible linkers include Gly-Ser linkers such as GGSGGGSGGSG (SEQ ID NO: 142), GGSGGGSGSG (SEQ ID NO: 143) and GGSGGGSG (SEQ ID NO: 144). Additional examples include: GGGGSGGGGS (SEQ ID NO: 145); GGGSGGGS (SEQ ID NO: 146); and GGSGGS (SEQ ID NO: 147). Linkers that include one or more antibody hinge regions and/or immunoglobulin heavy chain constant regions, such as CH3 alone or a CH2CH3 sequence can also be used.
In some situations, flexible linkers may be incapable of maintaining a distance or positioning of binding domains needed for a particular use. In these instances, rigid or semi-rigid linkers may be useful. Examples of rigid or semi-rigid linkers include proline-rich linkers. In particular embodiments, a proline-rich linker is a peptide sequence having more proline residues than would be expected based on chance alone. In particular embodiments, a proline-rich linker is one having at least 30%, at least 35%, at least 36%, at least 39%, at least 40%, at least 48%, at least 50%, or at least 51% proline residues. Particular examples of proline-rich linkers include fragments of proline-rich salivary proteins (PRPs).
It will also be understood by one of ordinary skill in the art that antibodies may undergo a variety of posttranslational modifications. The type and extent of these modifications often depends on the host cell line used to express the antibody as well as the culture conditions. Such modifications may include variations in glycosylation, methionine oxidation, diketopiperazine formation, aspartate isomerization and asparagine deamidation.
A monoclonal antibody includes an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies including the population are identical and/or bind the same epitope, except for possible variants that may arise during production of the monoclonal antibody, such variants generally being present in minor amounts. In contrast to polyclonal antibody preparations that typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. This type of antibody is produced by the daughter cells of a single antibody-producing hybridoma. A monoclonal antibody typically displays a single binding affinity for any epitope with which it binds.
The modifier “monoclonal” indicates the character of the antibody as being obtained from a homogeneous population of antibodies and is not to be construed as requiring production of the antibody by any particular method. Monoclonal antibodies recognize only one type of antigen. The monoclonal antibodies herein include “chimeric” antibodies (immunoglobulins) in which a portion of the heavy and/or light chain is identical with or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical with or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies. Techniques for the production of antibodies are well known in the art and described in, e.g., Harlow and Lane “Antibodies, A Laboratory Manual”, Cold Spring Harbor Laboratory Press, 1988; Harlow and Lane “Using Antibodies: A Laboratory Manual” Cold Spring Harbor Laboratory Press, 1999; Tickle et al. JALA: Journal of the Association for Laboratory Automation. 2009; 14(5): 303-307; Babcook et al. Proc. Natl. Acad. Sci. U.S.A. 1996; 93: 7843-7848; and U.S. Pat. No. 5,627,052.
In particular embodiments “affinity” refers to the strength of the sum total of noncovalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless indicated otherwise, as used herein, “binding affinity” refers to intrinsic binding affinity which reflects a 1:1 interaction between members of a binding pair (e.g., antibody and peptide). The affinity of a molecule X for its partner Y can generally be represented by the dissociation constant (K_D) or the association constant (K_A). Affinity can be measured by common methods known in the art.
In particular embodiments, “bind” means that the binding domain of an antibody associates with its target peptide with a dissociation constant (K_D) of 10⁻⁸M or less, in particular embodiments of from 10⁻⁵M to 10⁻¹³M, in particular embodiments of from 10⁻⁵M to 10⁻¹⁰M, in particular embodiments of from 10⁻⁵M to 10⁻⁷M, in particular embodiments of from 10⁻⁸M to 10⁻¹³M, or in particular embodiments of from 10⁻⁹M to 10⁻¹³M. The term can be further used to indicate that the binding domain does not bind to other biomolecules present, (e.g., it binds to other biomolecules with a dissociation constant (K_D) of 10⁻⁴M or more, in particular embodiments of from 10⁻⁴M to 1 M).
In particular embodiments, “bind” means that the binding domain of an antibody associates with its target peptide with an affinity constant (i.e., association constant, K_A) of 10⁷M⁻¹or more, in particular embodiments of from 10⁵M⁻¹to 10¹³M⁻¹, in particular embodiments of from 10⁵M⁻¹to 10¹⁰M⁻¹, in particular embodiments of from 10⁵M⁻¹to 10⁸M⁻¹, in particular embodiments of from 10⁷M⁻¹to 10¹³M⁻¹, or in particular embodiments of from 10⁷M⁻¹to 10⁸M⁻¹. The term can be further used to indicate that the binding domain does not bind to other biomolecules present, (e.g., it binds to other biomolecules with an association constant (K_A) of 10⁴M⁻¹or less, in particular embodiments of from 10⁴M⁻¹to 1 M⁻¹).
Antibodies of the present disclosure can be used for immunoaffinity enrichment of peptides described herein detected in SRM assays for diagnosis of SCID, WAS, XLA, cystinosis, and WD. Particular embodiments of the high affinity antibodies include anti-CD3ε 197, anti-WASp 274, anti-WASp 289, anti-BTK 407, anti-BTK 545, anti-CTNS 115, anti-CTNS 120, anti-CTNS 194, anti-CTNS 360, anti-SHPK 44, anti-SHPK 363, anti-SHPK 388, anti-ATP7B 214, anti-ATP7B 325, anti-ATP7B 466, anti-ATP7B 589, anti-ATP7B 621, anti-ATP7B 887, anti-ATP7B 1056, and anti-ATP7B 1061.
In particular embodiments, the exemplary antibodies include the CDRs presented in Table 2.

TABLE 2

Exemplary anti-peptide antibody CDRs of the present disclosure:

Antibody/
Disease	VH CDRs	VL CDRs

Anti-CD3ϵ	CDR1: EYVIH (SEQ ID NO: 22)	CDR1: RSSQSIVHSSGNTYLE (SEQ
197/SCID	CDR2: GFNPNIGGTNYNQRFKG	ID NO: 25)
	(SEQ ID NO: 23)	CDR2: KVSNRFS (SEQ ID NO: 26)
	CDR3: GGPYYYAMDY (SEQ ID NO:	CDR3: FQGSHVPWT (SEQ ID NO:
	24)	27)

Anti-WASp	CDR1: TYAMT (SEQ ID NO: 28)	CDR1: QSSETVYKNNYLA (SEQ ID
274/WAS	CDR2: SFYIEGSASYANWANGR	NO: 31)
	(SEQ ID NO: 29)	CDR2: WASKLAS (SEQ ID NO: 32)
	CDR3: GNPGGSSAV (SEQ ID NO: 30)	CDR3: AGYQSNIVDGTA (SEQ ID NO:
		33)

Anti-BTK	CDR1: RNEIS (SEQ ID NO: 34)	CDR1: QSSQSVYNNNRLA (SEQ ID
407/XLA	CDR2: GIGSPGRAYYATWAKSR	NO: 36)
	(SEQ ID NO: 35)	CDR2: SASTLAS (SEQ ID NO: 37)
	CDR3: GDI	CDR3: LGSYDCSTADCNA (SEQ ID
		NO: 38)

Anti-CTNS	CDR1: TRDGVC (SEQ ID NO: 39)	CDR1: QASQSIGSDLS (SEQ ID NO:
115/cystinosis		CDR2: CIYRGISATTSYASWAKGR 42)
	(SEQ ID NO: 40)	CDR2: KASKVET (SEQ ID NO: 43)
	CDR3: AWDL (SEQ ID NO: 41)	CDR3: QSIDFSKSYIGGA (SEQ ID
		NO: 44)

Anti-CTNS	CDR1: NNDGIC (SEQ ID NO: 45)	CDR1: QASQSIWNNNFLS (SEQ ID
360/cystinosis	CDR2: CIGSTSGRIYYASWAKGR	NO: 48)
	(SEQ ID NO: 46)	CDR2: EASKLAS (SEQ ID NO: 49)
	CDR3: EPYGSGSMAFDL (SEQ ID	CDR3: QGEFSCSIADCVA (SEQ ID
	NO: 47)	NO: 50)

Anti-SHPK	CDR1: SNYFMC (SEQ ID NO: 51)	CDR1: QASQSVYNNNDLA (SEQ ID
363/cystinosis	CDR2: CILVGSGRTTFASWAKGR	NO: 54)
	(SEQ ID NO: 52)	CDR2: GASTLVS (SEQ ID NO: 55)
	CDR3: AWAL (SEQ ID NO: 53)	CDR3: QGGYDPRNYP (SEQ ID NO:
		56)

Anti-ATP7B	CDR1: SYWII (SEQ ID NO: 57)	CDR1: QSSPSVANNNWLS (SEQ ID
1056/WD	CDR2: SSGPSGSAYYTSVVVKG	NO: 60)
	(SEQ ID NO: 58)	CDR2: GASTLAS (SEQ ID NO: 61)
	CDR3: AGGSDYDWFDL (SEQ ID NO:	CDR3: AGGHKTAEKNP (SEQ ID NO:
	59)	62)

In particular embodiments, the exemplary antibodies include the variable heavy and variable light domains presented in Table 3.

TABLE 3

Exemplary anti-peptide antibody variable heavy and variable
light domains of the present disclosure:

Antibody/
Disease	VH Domain	VL Domain

Anti-CD3ϵ	EVQLQQSGPDLVKPGASVKISCKT	DVLMTQNPLSLPVSLGDQASISCR
197/SCID	SGYIFIEYVIHWVKQSHGKSLEWIG	SSQSIVHSSGNTYLEWYLQKPGQ
	GFNPNIGGTNYNQRFKGKATLTVD	SPKVLIYKVSNRFSGVPDRFSGSG
	KSSSTAYMELRSLTSEDSAVYYCV	SGTDFTLKISRVEAEDLGVYYCFQ
	RGGPYYYAMDYWGQGTSVTVSS	GSHVPWTFGGGTKLEIK (SEQ ID
	(SEQ ID NO: 63)	NO: 64)

Anti-WASp	QSVEESGGRLVTPGTPLTLTCTVS	IVLTQTPASVSAAVGGTVTISCQS
274/WAS	GFSLSTYAMTWVRQAPGKGLQWI	SETVYKNNYLAWYQQKLGQPPKL
	GSFYIEGSASYANWANGRFTISKTS	LIYWASKLASGVPSRFKGSGSGT
	STVNLKMTSPTVADTASYFCARGN	QFTLTISDVVCADAGTYYCAGYQS
	PGGSSAVWGQGTLVTVSS (SEQ	NIVDGTAFGGGTEVVVN (SEQ ID
	ID NO: 65)	NO: 66)

Anti-BTK	QSVKESEGGLFKPTDTLTVTCTVS	QVLTQTVSPVSAAVGSTVTINCQS
407/XLA	GFSLSRNEISWFRQAPGNGLEWIG	SQSVYNNNRLAVVYQQKPGQPPK
	GIGSPGRAYYATWAKSRSTITRNT	GLIYSASTLASGVSSRFKGSGSGT
	NLNTVTLKMTSLTAADTATYFCAR	QFTLTISDVQCDDAATYYCLGSYD
	GDIWGPGTVVTVSS (SEQ ID NO:	CSTADCNAFGGGTEVVVK (SEQ
	67)	ID NO: 68)

Anti-CTNS	QEQLVESGGGLVQPEGSLTLTCKA	DVVMTQTPASVSEPVGGTVTIKC
115/cystinosis	SGFSFSTRDGVCVVVRQAPGKGLE	QASQSIGSDLSWYQQKPGQPPKR
	WIACIYRGISATTSYASWAKGRFTI	LIYKASKVETGVPSRFSGSGSGTE
	SKTSSTTVTLQMTSLTAADTATYFC	FTLTISDLECADASTYYCQSIDFSK
	ARAWDLWGPGTLVTVSS (SEQ ID	SYIGGAFGGGTEVVVK (SEQ ID
	NO: 69)	NO: 70)

Anti-CTNS	QSLEESGGGLFQPGASLTLTCTAS	QVLTQTASSVSAAVGGTVTVNCQ
360/cystinosis	GFSFSNNDGICWVRQAPGKGLEWI	ASQSIWNNNFLSWYQQKPGQPPK
	GCIGSTSGRIYYASWAKGRLTISKT	LLIYEASKLASGVPSRFSGSGSGT
	SSTTVILQMTSLTAADTATYFCASE	QFTLTISGVQCDDAATYYCQGEFS
	PYGSGSMAFDLWGPGTLVTVSS	CSIADCVAFGGGTEVVVR (SEQ ID
	(SEQ ID NO: 71)	NO: 72)

Anti-SHPK	QEQIEESGGGLVKPEGSLTLTCKV	QVLTQTPSPVSAAVGGTVTISCQA
363/cystinosis	SGFDFSSNYFMCWVRQAPGKGLE	SQSVYNNNDLAWYQQKPGQPPK
	WIGCILVGSGRTTFASWAKGRFTIS	LLIYGASTLVSGVPSRFSGSGSGA
	KTSSTTVTLQMTSLTAADTATYFCA	QFTLTISDLECDDAATYYCQGGYD
	RAWALWGPGTLVTVSS (SEQ ID	PRNYPFGGGTEVVVK (SEQ ID
	NO: 73)	NO: 74)

Anti-ATP7B	QSLEESGGRLVTPGGSLTVTCTVS	QVLTQTPSPVSAAVGGTVTINCQS
1056/WD	GFSLSSYWIIWVRQAPGEGLEWIG	SPSVANNNWLSWFQQKPGQRPK
	SSGPSGSAYYTSWVKGRFTISKTS	LLIYGASTLASGVPSRFKGSGSGT
	TTVDLKMTGLTTEDTATYFCARAG	QFTLTISDVQCDDAATYYCAGGHK
	GSDYDWFDLWGQGTLVTVSS	TAEKNPFGGGTEVVVK (SEQ ID
	(SEQ ID NO: 75)	NO: 76)

In particular embodiments, amino acid sequences of exemplary antibodies include: Anti-CD3ε 197 heavy chain variable domain amino acid sequence with leader sequence (SEQ ID NO: 79); Anti-CD3ε 197 light chain variable domain amino acid sequence with leader sequence (SEQ ID NO: 81); Anti-WASp 274 heavy chain amino acid sequence with leader sequence (SEQ ID NO: 83); Anti-WASp 274 heavy chain variable domain amino acid sequence with leader sequence (SEQ ID NO: 85); Anti-WASp 274 heavy chain amino acid sequence without leader sequence (SEQ ID NO: 86); Anti-WASp 274 light chain amino acid sequence with leader sequence (SEQ ID NO: 88); Anti-WASp 274 light chain variable domain amino acid sequence with leader sequence (SEQ ID NO: 90); Anti-WASp 274 light chain amino acid sequence without leader sequence (SEQ ID NO: 91); Anti-BTK 407 heavy chain amino acid sequence with leader sequence (SEQ ID NO: 93); Anti-BTK 407 heavy chain variable domain amino acid sequence with leader sequence (SEQ ID NO: 95); Anti-BTK 407 heavy chain amino acid sequence without leader sequence (SEQ ID NO: 96); Anti-BTK 407 light chain amino acid sequence with leader sequence (SEQ ID NO: 98); Anti-BTK 407 light chain variable domain amino acid sequence with leader sequence (SEQ ID NO: 100); Anti-BTK 407 light chain amino acid sequence without leader sequence (SEQ ID NO: 101); Anti-CTNS 115 heavy chain amino acid sequence with leader sequence (SEQ ID NO: 103); Anti-CTNS 115 heavy chain variable domain amino acid sequence with leader sequence (SEQ ID NO: 105); Anti-CTNS 115 heavy chain amino acid sequence without leader sequence (SEQ ID NO: 106); Anti-CTNS 115 light chain amino acid sequence with leader sequence (SEQ ID NO: 108); Anti-CTNS 115 light chain variable domain amino acid sequence with leader sequence (SEQ ID NO: 110); Anti-CTNS 115 light chain amino acid sequence without leader sequence (SEQ ID NO: 111); Anti-CTNS 360 heavy chain amino acid sequence with leader sequence (SEQ ID NO: 113); Anti-CTNS 360 heavy chain variable domain amino acid sequence with leader sequence (SEQ ID NO: 115); Anti-CTNS 360 heavy chain amino acid sequence without leader sequence (SEQ ID NO: 116); Anti-CTNS 360 light chain amino acid sequence with leader sequence (SEQ ID NO: 118); Anti-CTNS 360 light chain variable domain amino acid sequence with leader sequence (SEQ ID NO: 120); Anti-CTNS 360 light chain amino acid sequence without leader sequence (SEQ ID NO: 121); Anti-SHPK 363 heavy chain amino acid sequence with leader sequence (SEQ ID NO: 123); Anti-SHPK 363 heavy chain variable domain amino acid sequence with leader sequence (SEQ ID NO: 125); Anti-SHPK 363 heavy chain amino acid sequence without leader sequence (SEQ ID NO: 126); Anti-SHPK 363 light chain amino acid sequence with leader sequence (SEQ ID NO: 128); Anti-SHPK 363 light chain variable domain amino acid sequence with leader sequence (SEQ ID NO: 130); Anti-SHPK 363 light chain amino acid sequence without leader sequence (SEQ ID NO: 131); Anti-ATP7B 1056 heavy chain amino acid sequence with leader sequence (SEQ ID NO: 133); Anti-ATP7B 1056 heavy chain variable domain amino acid sequence with leader sequence (SEQ ID NO: 135); Anti-ATP7B 1056 heavy chain amino acid sequence without leader sequence (SEQ ID NO: 136); Anti-ATP7B 1056 light chain amino acid sequence with leader sequence (SEQ ID NO: 138); Anti-ATP7B 1056 light chain variable domain amino acid sequence with leader sequence (SEQ ID NO: 140); and Anti-ATP7B 1056 light chain amino acid sequence without leader sequence (SEQ ID NO: 141).
In particular embodiments, coding sequences of exemplary antibodies include: Anti-CD3ε 197 heavy chain variable domain coding sequence with leader sequence (SEQ ID NO: 78); Anti-CD3ε 197 light chain variable domain coding sequence with leader sequence (SEQ ID NO: 80); Anti-WASp 274 heavy chain coding sequence (EB0603-2F8-H2) with leader sequence (SEQ ID NO: 82); Anti-WASp 274 heavy chain variable domain coding sequence with leader sequence (SEQ ID NO: 84); Anti-WASp 274 light chain coding sequence (EB0603-2F8-K2) with leader sequence (SEQ ID NO: 87); Anti-WASp 274 light chain variable domain coding sequence with leader sequence (SEQ ID NO: 89); Anti-BTK 407 heavy chain coding sequence (EB0602-1G5-H2) with leader sequence (SEQ ID NO: 92); Anti-BTK 407 heavy chain variable domain coding sequence with leader sequence (SEQ ID NO: 94); Anti-BTK 407 light chain coding sequence (EB0602-1G5-K1) with leader sequence (SEQ ID NO: 97); Anti-BTK 407 light chain variable domain coding sequence with leader sequence (SEQ ID NO: 99); Anti-CTNS 115 heavy chain coding sequence (EB0606-3H8-H5) with leader sequence (SEQ ID NO: 102); Anti-CTNS 115 heavy chain variable domain coding sequence with leader sequence (SEQ ID NO: 104); Anti-CTNS 115 light chain coding sequence (EB0606-3H8-K7) with leader sequence (SEQ ID NO: 107); Anti-CTNS 115 light chain variable domain coding sequence with leader sequence (SEQ ID NO: 109); Anti-CTNS 360 heavy chain coding sequence (EB0604-4E3-H4) with leader sequence (SEQ ID NO: 112); Anti-CTNS 360 heavy chain variable domain coding sequence with leader sequence (SEQ ID NO: 114); Anti-CTNS 360 light chain coding sequence (EB0604-4E3-K1) with leader sequence (SEQ ID NO: 117); Anti-CTNS 360 light chain variable domain coding sequence with leader sequence (SEQ ID NO: 119); Anti-SHPK 363 heavy chain coding sequence (EB0605B-6G12-H1) with leader sequence (SEQ ID NO: 122); Anti-SHPK 363 heavy chain variable domain coding sequence with leader sequence (SEQ ID NO: 124); Anti-SHPK 363 light chain coding sequence (EB0605B-6G12-K1) with leader sequence (SEQ ID NO: 127); Anti-SHPK 363 light chain variable domain coding sequence with leader sequence (SEQ ID NO: 129); Anti-ATP7B 1056 heavy chain coding sequence (EB0601-H3-1) with leader sequence (SEQ ID NO: 132); Anti-ATP7B 1056 heavy chain variable domain coding sequence with leader sequence (SEQ ID NO: 134); Anti-ATP7B 1056 light chain coding sequence (EB0601-K3-2) with leader sequence (SEQ ID NO: 137); and Anti-ATP7B 1056 light chain variable domain coding sequence with leader sequence (SEQ ID NO: 139).

(V) ENRICHMENT STRATEGIES FOR PEPTIDES

Enrichment of a desired peptide target prior to SRM can be accomplished by any means known in the art. A host of enrichment procedures are available, including immuno adsorption-based depletion of abundant protein species from samples, precipitation, chromatography, electrophoresis, solvent partitioning, immunoprecipitation, immunoelectrophoresis, and immunochromatography. In particular embodiments, a SISCAPA method for specific antibody-based capture of individual tryptic peptides from a digest of a sample can be used. Anderson et al., J. Proteome Research 2004; 3: 235-244; U.S. Pat. No. 7,632,686.
In particular embodiments, the antibodies that bind the peptide markers, such as the antibodies disclosed herein, can be attached to a solid support. Particular embodiments use an affinity column, where antibodies are covalently coupled to chromatography media. In particular embodiments, POROS (Applied Biosystems, Foster City, Calif.) nanocolumns can be used in SISCAPA enrichment and features high binding capacity, a relatively high concentration of antibodies allowing for rapid enrichment of target peptides, and the ability to prepare columns with a variety of functionalized groups. Alternatively, antibodies can be attached to beads, magnetic beads, or other solid particle. One means of attachment is conjugation of the antibody to a protein coated on the beads. For example, Protein G coated particles offer the binding of antibodies in a preferred orientation. Other means of attachment can be used, such as direct coating of a bead with the antibody. Magnetic particles are available in a wide array of chemistries allowing for coupling to antibodies. Enrichment with antibodies attached to particles can allow parallel processing of samples. Magnetic particle processing has been automated in 96 well plates for the SISCAPA enrichment step with elution in the plates for analysis by mass spectrometry. Other particular embodiments use a novel bead trap device developed to perform the bead handling steps in line with a nanoflow chromatography system. Anderson et al. Mol Cell Proteomics 2009; 8(5): 995-1005. This minimizes losses of peptides to containers between elution and analysis steps. Peptide enrichment can also be implemented by immobilizing anti-peptide antibodies in pipet tips. Nelson et al. Anal Chem. 1995; 67(7): 1153-1158. After separation of the antibody bound peptide from free peptides, the bound peptide can be eluted. Any elution means can be used. One elution means which has been found to be efficient is 5% acetic acid/3% acetonitrile. Other elution means, including other acids, and other concentrations of acetic acid can be used, as is efficient for a particular peptide.

(VI) LIQUID CHROMATOGRAPHY (LC)

In particular embodiments, one or more LC purification steps are performed prior to SRM-MS. A mixture of enriched peptides (the mobile phase) can be passed through a column packed with material (stationary phase) to separate the peptides based on their weight and affinity for the mobile and stationary phases of the column. Traditional LC analysis relies on the chemical interactions between sample components and column packing materials, where laminar flow of the sample through the column is the basis for separation of the analyte of interest from the test sample. The skilled artisan will understand that separation in such columns is a diffusional process. A variety of column packing materials are available for chromatographic separation of samples, and selection of an appropriate separation protocol is an empirical process that depends on the sample characteristics, the analyte of interest, the interfering substances present and their characteristics, etc. Various packing chemistries can be used depending on the needs (e.g., structure, polarity, and solubility of compounds being purified). In particular embodiments the columns are polar, ion exchange (both cation and anion), hydrophobic interaction, phenyl, C-2, C-8, C-18 columns, polar coating on porous polymer, or others that are commercially available. During chromatography, the separation of materials is affected by variables such as choice of eluant (also known as a “mobile phase”), choice of gradient elution and the gradient conditions, temperature, etc. In particular embodiments, an analyte may be purified by applying a sample to a column under conditions where the analyte of interest is reversibly retained by the column packing material, while one or more other materials are not retained. In these embodiments, a first mobile phase condition can be employed where the analyte of interest is retained by the column, and a second mobile phase condition can subsequently be employed to remove retained material from the column, once the non-retained materials are washed through. Alternatively, an analyte may be purified by applying a sample to a column under mobile phase conditions where the analyte of interest elutes at a differential rate in comparison to one or more other materials. As discussed above, such procedures may enrich the amount of one or more analytes of interest relative to one or more other components of the sample. In particular embodiments, the LC is nanoflow LC (nanoLC). In nanoflow LC (nanoLC) chromatographic separations are performed using flow rates in the range of low nanoliter per minute, which result in high analytical sensitivity due to the large concentration efficiency afforded by this type of chromatography. Cutillas, Current Nanoscience, 2005; 1: 65-71.

(VII) MASS SPECTROMETRY (MS)

A mass spectrometer includes a gas phase ion spectrometer that measures a parameter that can be translated into mass-to-charge (m/z) ratios of gas phase ions. Mass spectrometry refers to the use of a mass spectrometer to detect gas phase ions. Mass spectrometers generally include an ion source and a mass analyzer. Examples of mass spectrometers are time-of-flight (TOF), magnetic sector, quadrupole filter, ion trap, ion cyclotron resonance, electrostatic sector analyzer and hybrids of these. A laser desorption mass spectrometer includes a mass spectrometer that uses laser energy as a means to desorb, volatilize, and ionize an analyte. A tandem mass spectrometer includes any mass spectrometer that is capable of performing two successive stages of m/z-based discrimination or measurement of ions, including ions in an ion mixture. The phrase includes mass spectrometers having two mass analyzers that are capable of performing two successive stages of m/z-based discrimination or measurement of ions tandem-in-space. The phrase further includes mass spectrometers having a single mass analyzer that is capable of performing two successive stages of m/z-based discrimination or measurement of ions tandem-in-time. The phrase thus explicitly includes Qq-TOF mass spectrometers, ion trap mass spectrometers, ion trap-TOF mass spectrometers, TOF-TOF mass spectrometers, Fourier transform ion cyclotron resonance mass spectrometers, electrostatic sector-magnetic sector mass spectrometers, and combinations thereof.
Ionization in mass spectrometry includes the process by which analytes in a sample are ionized. Such analytes may become charged molecules used for further analysis. For example, sample ionization may be performed by electrospray ionization (ESI), laserspray ionization (LSI) atmospheric pressure chemical ionization (APCI), photoionization, electron ionization, fast atom bombardment (FAB)/liquid secondary ionization (LSIMS), matrix assisted laser desorption ionization (MALDI), field ionization, field desorption, thermospray/plasmaspray ionization, and particle beam ionization. The skilled artisan will understand that the choice of ionization method can be determined based on the analyte to be measured, type of sample, the type of detector, the choice of positive versus negative mode, etc.
A mass analyzer includes the component of the mass spectrometer that takes ionized masses and separates them based on m/z ratios and outputs them to the detector where they are detected and later converted to a digital output. Suitable mass analyzers for determining m/z ratios include quadrupole mass analyzer, time-of-flight (TOF) mass analyzer, magnetic or electrostatic sector mass analyzer and ion trap (e.g. ion cyclotron resonance) mass analyzer.
A selected reaction monitoring (SRM)-MS assay targets a predetermined set of peptides for a given protein of interest. SRM is a tandem mass spectrometry mode in which an ion of a particular mass, the parent or precursor ion, is selected in the first stage of tandem mass spectrometry, and an ion product of a fragmentation reaction of the precursor ion is selected in the second mass spectrometry stage for detection. The specific pair of m/z values associated with a selected precursor ion and fragment ion is referred to as a transition. For each signature peptide, those fragment ions that provide optimal signal intensity and discriminate the targeted peptide from other species present in the sample are identified. Optimized transitions contribute to an effective SRM assay. Several such transitions (precursor/fragment ion pairs) are monitored over time, yielding a set of chromatographic traces with the retention time and signal intensity for a specific transition as coordinates. SRM-MS analysis of signature peptides are generally performed on a triple quadrupole mass spectrometer (QQQ-MS), an instrument with the capability to selectively isolate precursor ions corresponding to the m/z of the signature peptides and to selectively monitor peptide-specific fragment ions. In SRM analysis, the specificity depends on multiple mass analyzers (mass filters). The first quadrupole is to select the desired parent or precursor ion. The third quadrupole is to monitor the (one or more) fragment ion(s). The fragment ion(s) is generated through collisional induced dissociation in the second quadrupole. The two levels of mass selection allow high selectivity, as co-eluting background ions are filtered out very effectively. Unlike conventional tandem mass spectrometry (MS/MS) experiments that survey all analytes in a sample, SRM analysis selectively targets (filters) particular analytes, which translates into an increased sensitivity by one or two orders of magnitude compared with conventional ‘full scan’ techniques. In addition, SRM provides a linear response over a wide dynamic range up to five orders of magnitude. This enables the detection of low-abundance proteins in highly complex mixtures. Therefore, SRM is a highly specific detection/monitoring method with low background interference. When multiple parent ions are monitored in a single MS run, this type of analysis is known as multiple reaction monitoring (MRM). Using MRM analysis, multiple proteins and multiple regions (signature peptides) of a protein can be monitored in a single mass spectrometry run. Selected reaction monitoring/multiple reaction monitoring mass spectrometry (SRM/MRM-MS) is described in, e.g., U.S. Pat. No. 8,383,417, WO 2013/106603, and US 2013/105684.
In particular embodiments, the following parameters can be used to specify an LC-SRM-MS assay of a protein under a particular LC-SRM-MS system: (1) an enriched for tryptic peptide of a given protein; (2) the retention time (RT) of the peptide on an LC column; (3) the m/z value of the peptide precursor ion; (4) the declustering potential used to ionize the precursor ion; (5) the m/z value of a fragment ion generated from the peptide precursor ion; and (6) the collision energy (CE) used to fragment the peptide precursor ion that is optimized for the particular peptide. RT includes the elapsed time between injection and elution of an analyte. Declustering potential (DP) includes a voltage potential to dissolvate and dissociate ion clusters. It is also known as “fragmentor voltage” or “ion transfer capillary offset voltage” depending on the manufacturer. Collision energy (CE) includes the amount of energy precursor ions receive as they are accelerated into the collision cell.
To facilitate accurate quantification of the peptides by the methods disclosed herein, a set of isotopically-labeled synthetic versions of the peptides of interest may be added in known amounts to the sample for use as internal standards. Since the isotopically-labeled peptides have physical and chemical properties identical to the corresponding surrogate peptide, they co-elute from the chromatographic column and are easily identifiable on the resultant mass spectrum. Gerber et al. Proc. Natl. Asso. Sci. 2003; 100: 6940-6945; Kirkpatrick et al. Methods 2005; 35: 265-273. The isotopes with which amino acids in a given peptide can be labeled include ¹³C, ²H, ¹⁵N, ¹⁷O, ¹⁸O, and ³⁴S. In particular embodiments, a peptide is labeled with ¹³C and/or ¹⁵N heavy isotopes. The addition of the labeled standards may occur before or after proteolytic digestion. In particular embodiments, the labeled internal standard peptides are added prior to an LC-MRM-MS assay. Methods of synthesizing isotopically-labeled peptides will be known to those of skill in the art. Thus, in particular embodiments, the experimental samples contain internal standard peptides. In particular embodiments, internal standard peptides include reference signature peptides. In particular embodiments, a signature peptide concentration can be determined by combining: (i) a ratio calculated from comparing the peak area of the signature peptide to the peak area of its corresponding reference signature peptide obtained from an LC-MRM-MS assay, and (ii) the known concentration of the reference signature peptide. Peptides selected as reference standards and suitable for quantification are sometimes referred to as quantotypic peptides (Q-peptides). Q-peptides include all of the characteristics of proteotypic peptides but also place restrictions on the residues that can constitute the reference peptide to eradicate artefactual modification and/or incomplete cleavage. Holman et al. Bioanalysis 2012; 4(14): 1763-1786.
Absolute quantitative levels of a given protein, or proteins, can be determined by the SRM/MRM methodology whereby the SRM/MRM signature peak area of an individual peptide from a given protein in one biological sample is compared to the SRM/MRM signature peak area of a known amount of a “spiked” internal standard. In particular embodiments, the internal standard is a synthetic version of the same exact peptide that contains one or more amino acid residues labeled with one or more heavy isotopes. Such isotope labeled internal standards are synthesized so that mass spectrometry analysis generates a predictable and consistent SRM/MRM signature peak that is different and distinct from the native peptide signature peak, and which can be used as a comparator peak. Thus, when the internal standard is spiked in known amounts into a protein preparation from a biological sample and analyzed by mass spectrometry, the signature peak area of the native peptide is compared to the signature peak area of the internal standard peptide, and this numerical comparison indicates either the absolute molarity and/or absolute weight of the native peptide present in the original protein preparation from the biological sample. Absolute quantitative data for fragment peptides are displayed according to the amount of protein analyzed per sample. Absolute quantitation can be performed across many peptides, and thus proteins, simultaneously in a single sample and/or across many samples to gain insight into absolute protein amounts in individual biological samples and in entire cohorts of individual samples.
Another strategy for absolute quantitation of peptides is equimolarity through equalizer peptide. This methodology involves chemically synthesizing the isotopically labeled Q-peptides of interest as dipeptides. A common amino acid sequence is positioned N-terminal to the Q-peptide and is referred to as the equalizer peptide. After solubilization and proteolytic digestion, the amount of Q-peptide can be accurately determined through reference to a single light-labeled peptide. Appropriate amounts of each standard peptide can then be added to a sample of interest (either predigested or prior to proteolysis) to facilitate absolute quantification. Holzmann et al. Anal. Chem. 2009; 81: 10254-10261. Absolute quantification can also employ quantification concatemer (QconCAT) proteins. Beynon et al. Nat. Methods 2005; 2: 587-589; Johnson et al. J. Am. Soc. Mass Spectrom. 2009; 20: 2211-2220; Ding et al. J. Proteome Res. 2011; 10: 3652-3659; Carroll et al. Molecular & Cellular Proteomics 2011; September 19: mcp-M111. In this strategy, a recombinant artificial protein that is an affinity tagged, concatenation of standard peptides from several proteins of interest is heterologously produced in Escherichia coli grown in stable isotopically enriched media. The QconCAT protein is then affinity purified and co-digested with the sample, generating a stoichiometric mixture of all the ‘heavy’ Q-peptides of which it is composed, and the proteolytic peptides from the native proteins and internal standard are subsequently analyzed. A variant of the QconCAT approach, termed peptide-concatenated standards (PCS), uses flanking regions between the Q-peptides in the artificial protein sequence that mirror their endogenous environment. Kito et al. J. Proteome Res. 2007; 6: 792-800. Other particular embodiments use protein standards for absolute quantification (PSAQ). Brun et al. Mol. Cell. Proteomics 2007; 6: 2139-2149. PSAQ uses recombinant proteins but rather than being a concatenation of peptides from several proteins, the entire protein to be quantified is expressed in stable isotope-labeled form. One or several PSAQs can then be added to the sample pre-digestion to facilitate quantification.
Particular embodiments use label-free strategies for protein quantification such as intensity based measurements (America and Cordewener, Proteomics 2008; 8: 731-749) or spectral counting (Lundgren et al. Expert Rev. Proteomics 2010; 7: 39-53).
To obtain relative quantitative levels of a given peptide, the mass spectrometry-derived signature peak area (or the peak height if the peaks are sufficiently resolved) of an individual peptide, or multiple peptides, from a given protein, in one biological sample can be compared to the signature peak area determined for the same peptide, or peptides, from the same protein, in one or more additional and different biological samples, using the same SRM/MRM methodology. In this way, the amount of a particular peptide, or peptides, from a given protein, is determined relative to the same peptide, or peptides, from the same protein across two or more biological samples under the same experimental conditions. In addition, relative quantitation can be determined for a given peptide, or peptides, from a single protein within a single sample by comparing the signature peak area for that peptide for that given protein by SRM/MRM methodology to the signature peak area for another and different peptide, or peptides, from a different protein within the same protein preparation from the biological sample. In this way, the amount of a particular peptide from a given protein, and therefore the amount of the given protein, is determined relative to another protein within the same sample. These approaches generate quantitation of an individual peptide, or peptides, from a given protein to the amount of another peptide, or peptides, from the same protein or from a different protein between samples and within samples wherein the amounts as determined by signature peak area are relative one to another, regardless of the absolute weight to volume or weight to weight amounts of peptides in the protein preparation from the biological sample. Relative quantitative data about individual signature peak areas between different samples can be normalized to the amount of protein analyzed per sample. Relative quantitation can be performed across many peptides simultaneously in a single sample and/or across many samples to gain insight into relative protein amounts.
Signature peptide levels can be expressed in concentration units (e.g., pmol/L). In particular embodiments, the mean concentration of a signature peptide in a test sample derived from a subject being screened for SCID, WAS, XLA, cystinosis, and/or WD can be compared to the mean concentration of the corresponding peptide in a normal control sample. In particular embodiments, a normal control sample can be derived from one or more normal control subjects or from a population of normal control subjects. In particular embodiments, a normal control subject includes a subject who does not have or is not known to have SCID, WAS, XLA, cystinosis, or WD. In particular embodiments, a normal control subject includes a subject who does not have genetic mutations associated with SCID, WAS, XLA, cystinosis, or WD.
In particular embodiments, the mean concentration of a CD3ε signature peptide in DBS from a population of normal control subjects includes a concentration in a range of 70 pmol/L to 400 pmol/L, in a range of 80 pmol/L to 300 pmol/L, and in a range of 90 pmol/L to 200 pmol/L. In particular embodiments, the mean concentration of a CD3ε signature peptide in DBS from a population of normal control subjects includes a concentration of 70 pmol/L, 80 pmol/L, 90 pmol/L, 100 pmol/L, 110 pmol/L, 120 pmol/L, 130 pmol/L, 140 pmol/L, 150 pmol/L, 160 pmol/L, 170 pmol/L, 180 pmol/L, 190 pmol/L, 200 pmol/L, 210 pmol/L, 220 pmol/L, 230 pmol/L, 240 pmol/L, 250 pmol/L, 260 pmol/L, 270 pmol/L, 280 pmol/L, 290 pmol/L, 300 pmol/L, 310 pmol/L, 320 pmol/L, 330 pmol/L, 340 pmol/L, 350 pmol/L, 360 pmol/L, 370 pmol/L, 380 pmol/L, 390 pmol/L, 400 pmol/L, or more.
In particular embodiments, the mean concentration of a WASp 274 signature peptide in DBS from a population of normal control subjects includes a concentration in a range of 600 pmol/L to 5000 pmol/L, in a range of 700 pmol/L to 3000 pmol/L, and in a range of 800 pmol/L to 2500 pmol/L. In particular embodiments, the mean concentration of a WASp 274 signature peptide in DBS from a population of normal control subjects includes a concentration of 600 pmol/L, 700 pmol/L, 800 pmol/L, 900 pmol/L, 1000 pmol/L, 1100 pmol/L, 1200 pmol/L, 1300 pmol/L, 1400 pmol/L, 1500 pmol/L, 1600 pmol/L, 1700 pmol/L, 1800 pmol/L, 1900 pmol/L, 2000 pmol/L, 2100 pmol/L, 2200 pmol/L, 2300 pmol/L, 2400 pmol/L, 2500 pmol/L, 2600 pmol/L, 2700 pmol/L, 2800 pmol/L, 2900 pmol/L, 3000 pmol/L, 3100 pmol/L, 3200 pmol/L, 3300 pmol/L, 3400 pmol/L, 3500 pmol/L, 3600 pmol/L, 3700 pmol/L, 3800 pmol/L, 3900 pmol/L, 4000 pmol/L, 4100 pmol/L, 4200 pmol/L, 4300 pmol/L, 4400 pmol/L, 4500 pmol/L, 4600 pmol/L, 4700 pmol/L, 4800 pmol/L, 4900 pmol/L, 5000 pmol/L, or more.
In particular embodiments, the mean concentration of a WASp 289 signature peptide in DBS from a population of normal control subjects includes a concentration in a range of 5500 pmol/L to 15000 pmol/L, in a range of 6000 pmol/L to 14000 pmol/L, and in a range of 6500 pmol/L to 13000 pmol/L. In particular embodiments, the mean concentration of a WASp 289 signature peptide in DBS from a population of normal control subjects includes a concentration of 5500 pmol/L, 5600 pmol/L, 5700 pmol/L, 5800 pmol/L, 5900 pmol/L, 6000 pmol/L, 6100 pmol/L, 6200 pmol/L, 6300 pmol/L, 6400 pmol/L, 6500 pmol/L, 6600 pmol/L, 6700 pmol/L, 6800 pmol/L, 6900 pmol/L, 7000 pmol/L, 7100 pmol/L, 7200 pmol/L, 7300 pmol/L, 7400 pmol/L, 7500 pmol/L, 7600 pmol/L, 7700 pmol/L, 7800 pmol/L, 7900 pmol/L, 8000 pmol/L, 8100 pmol/L, 8200 pmol/L, 8300 pmol/L, 8400 pmol/L, 8500 pmol/L, 8600 pmol/L, 8700 pmol/L, 8800 pmol/L, 8900 pmol/L, 9000 pmol/L, 9800 pmol/L, 9900 pmol/L, 10000 pmol/L, 10100 pmol/L, 10200 pmol/L, 10300 pmol/L, 10400 pmol/L, 10500 pmol/L, 10600 pmol/L, 10700 pmol/L, 10800 pmol/L, 10900 pmol/L, 11000 pmol/L, 11100 pmol/L, 11200 pmol/L, 11300 pmol/L, 11400 pmol/L, 11500 pmol/L, 11600 pmol/L, 11700 pmol/L, 11800 pmol/L, 11900 pmol/L, 12000 pmol/L, 12100 pmol/L, 12200 pmol/L, 12300 pmol/L, 12400 pmol/L, 12500 pmol/L, 12600 pmol/L, 12700 pmol/L, 12800 pmol/L, 12900 pmol/L, 13000 pmol/L, 13100 pmol/L, 13200 pmol/L, 113300 pmol/L, 13400 pmol/L, 13500 pmol/L, 13600 pmol/L, 13700 pmol/L, 13800 pmol/L, 13900 pmol/L, 14000 pmol/L, 14100 pmol/L, 14200 pmol/L, 14300 pmol/L, 14400 pmol/L, 14500 pmol/L, 14600 pmol/L, 14700 pmol/L, 14800 pmol/L, 14900 pmol/L, 15000 pmol/L, or more.
In particular embodiments, the mean concentration of a BTK 407 signature peptide in DBS from a population of normal control subjects includes a concentration in a range of 350 pmol/L to 2500 pmol/L, in a range of 450 pmol/L to 2400 pmol/L, and in a range of 550 pmol/L to 2300 pmol/L. In particular embodiments, the mean concentration of a BTK 407 signature peptide in DBS from a population of normal control subjects includes a concentration of 350 pmol/L, 400 pmol/L, 450 pmol/L, 500 pmol/L, 550 pmol/L, 600 pmol/L, 650 pmol/L, 700 pmol/L, 750 pmol/L, 800 pmol/L, 850 pmol/L, 900 pmol/L, 1000 pmol/L, 1100 pmol/L, 1200 pmol/L, 1300 pmol/L, 1400 pmol/L, 1500 pmol/L, 1600 pmol/L, 1700 pmol/L, 1800 pmol/L, 1900 pmol/L, 2000 pmol/L, 2100 pmol/L, 2200 pmol/L, 2300 pmol/L, 2400 pmol/L, 2500 pmol/L, or more.
In particular embodiments, the mean concentration of a BTK 545 signature peptide in DBS from a population of normal control subjects includes a concentration in a range of 550 pmol/L to 1600 pmol/L, in a range of 650 pmol/L to 1500 pmol/L, and in a range of 750 pmol/L to 1400 pmol/L. In particular embodiments, the mean concentration of a BTK 545 signature peptide in DBS from a population of normal control subjects includes a concentration of 550 pmol/L, 600 pmol/L, 650 pmol/L, 700 pmol/L, 750 pmol/L, 800 pmol/L, 850 pmol/L, 900 pmol/L, 950 pmol/L, 1000 pmol/L, 1050 pmol/L, 1100 pmol/L, 1150 pmol/L, 1200 pmol/L, 1250 pmol/L, 1300 pmol/L, 1350 pmol/L, 1400 pmol/L, 1450 pmol/L, 1500 pmol/L, 1550 pmol/L, 1600 pmol/L, or more.
In particular embodiments, the mean concentration of a CTNS 115 signature peptide in DBS from a population of normal control subjects includes a concentration in a range of 40 pmol/L to 250 pmol/L, in a range of 50 pmol/L to 200 pmol/L, and in a range of 60 pmol/L to 150 pmol/L. In particular embodiments, the mean concentration of a CTNS 115 signature peptide in DBS from a population of normal control subjects includes a concentration of 40 pmol/L, 45 pmol/L, 50 pmol/L, 55 pmol/L, 60 pmol/L, 65 pmol/L, 70 pmol/L, 75 pmol/L, 80 pmol/L, 85 pmol/L, 90 pmol/L, 95 pmol/L, 100 pmol/L, 110 pmol/L, 120 pmol/L, 130 pmol/L, 140 pmol/L, 150 pmol/L, 160 pmol/L, 170 pmol/L, 180 pmol/L, 190 pmol/L, 200 pmol/L, 210 pmol/L, 220 pmol/L, 230 pmol/L, 240 pmol/L, 250 pmol/L, or more.
In particular embodiments, the mean concentration of a SHPK 363 signature peptide in DBS from a population of normal control subjects includes a concentration in a range of 100 pmol/L to 8000 pmol/L, in a range of 300 pmol/L to 7000 pmol/L, and in a range of 500 pmol/L to 6000 pmol/L. In particular embodiments, the mean concentration of a SHPK 363 signature peptide in DBS from a population of normal control subjects includes a concentration of 100 pmol/L, 200 pmol/L, 300 pmol/L, 400 pmol/L, 500 pmol/L, 600 pmol/L, 700 pmol/L, 800 pmol/L, 900 pmol/L, 1000 pmol/L, 1500 pmol/L, 2000 pmol/L, 2500 pmol/L, 3000 pmol/L, 3500 pmol/L, 4000 pmol/L, 4500 pmol/L, 5000 pmol/L, 5500 pmol/L, 6000 pmol/L, 6500 pmol/L, 7000 pmol/L, 7500 pmol/L, 8000 pmol/L, or more.
In particular embodiments, the mean concentration of an ATP7B 214 signature peptide in DBS from a population of normal control subjects includes a concentration in a range of 30 pmol/L to 100 pmol/L, in a range of 40 pmol/L to 90 pmol/L, and in a range of 50 pmol/L to 80 pmol/L. In particular embodiments, the mean concentration of an ATP7B 214 signature peptide in DBS in a population of normal control subjects includes a concentration of 30 pmol/L, 35 pmol/L, 40 pmol/L, 45 pmol/L, 50 pmol/L, 55 pmol/L, 60 pmol/L, 65 pmol/L, 70 pmol/L, 75 pmol/L, 80 pmol/L, 85 pmol/L, 90 pmol/L, 95 pmol/L, 100 pmol/L, or more.
In particular embodiments, the mean concentration of an ATP7B 887 signature peptide in DBS from a population of normal control subjects includes a concentration in a range of 200 pmol/L to 500 pmol/L, in a range of 250 pmol/L to 450 pmol/L, and in a range of 300 pmol/L to 400 pmol/L. In particular embodiments, the mean concentration of an ATP7B 887 signature peptide in DBS from a population of normal control subjects includes a concentration of 200 pmol/L, 210 pmol/L, 220 pmol/L, 230 pmol/L, 240 pmol/L, 250 pmol/L, 260 pmol/L, 270 pmol/L, 280 pmol/L, 290 pmol/L, 300 pmol/L, 310 pmol/L, 320 pmol/L, 330 pmol/L, 340 pmol/L, 350 pmol/L, 360 pmol/L, 370 pmol/L, 380 pmol/L, 390 pmol/L, 400 pmol/L, 410 pmol/L, 420 pmol/L, 430 pmol/L, 440 pmol/L, 450 pmol/L, 460 pmol/L, 470 pmol/L, 480 pmol/L, 490 pmol/L, 500 pmol/L, or more.
In particular embodiments, the mean concentration of an ATP7B 1056 signature peptide in DBS from a population of normal control subjects includes a concentration in a range of 90 pmol/L to 400 pmol/L, in a range of 100 pmol/L to 300 pmol/L, and in a range of 150 pmol/L to 250 pmol/L. In particular embodiments, the mean concentration of an ATP7B 1056 signature peptide in DBS from a population of normal control subjects includes a concentration of 90 pmol/L, 100 pmol/L, 110 pmol/L, 120 pmol/L, 130 pmol/L, 140 pmol/L, 150 pmol/L, 160 pmol/L, 170 pmol/L, 180 pmol/L, 190 pmol/L, 200 pmol/L, 210 pmol/L, 220 pmol/L, 230 pmol/L, 240 pmol/L, 250 pmol/L, 260 pmol/L, 270 pmol/L, 280 pmol/L, 290 pmol/L, 300 pmol/L, 310 pmol/L, 320 pmol/L, 330 pmol/L, 340 pmol/L, 350 pmol/L, 360 pmol/L, 370 pmol/L, 380 pmol/L, 390 pmol/L, 400 pmol/L, or more.
One or more standard peptides may be synthesized with any method known in the pertinent art. Such synthetic peptides may further include amino acids with one or more natural modifications. Such natural modifications may include deamination of glutamine and asparagine, amination, oxidation, and hydroxylation.

(VIII) METHODS OF USE

The methods of the present disclosure include identifying individuals with one or more of SCID, WAS, XLA, cystinosis, and/or WD. In particular embodiments, diagnosing individuals with SCID, WAS, XLA, cystinosis, and/or WD is performed early, for example, as part of NBS, or before symptoms of a disorder are evident in the individual.
The methods of the present disclosure include obtaining DBS samples. In particular embodiments, DBS are obtained according to a method described above. In particular embodiments, DBS are obtained from a DBS repository or lab that stores DBS for future testing.
The methods of the present disclosure include digesting proteins in DBS with digestion enzymes. In particular embodiments, one or more punches of the DBS or the whole DBS can be solubilized in an appropriate buffer, and an appropriate digestion enzyme described above can be added to digest proteins present in DBS into peptide fragments. In particular embodiments, DBS can be solubilized with 0.1% ProteaseMax™ in 50 mM ammonium biocarbonate (pH 8) and digested with trypsin.
The methods of the present disclosure include enriching for signature peptides that are used in screening for SCID, WAS, XLA, cystinosis, and/or WD. Signature peptides include CD3ε 197 for SCID; WASp 274 for WAS; WASp 289 for WAS; BTK 407 for XLA; BTK 545 for XLA; CTNS 115 for cystinosis; CTNS 120 for cystinosis; CTNS 194 for cystinosis; CTNS 360 for cystinosis; SHPK 44 for cystinosis; SHPK 363 for cystinosis; SHPK 388 for cystinosis; ATP7B 214 for WD; ATP7B 325 for WD; ATP7B 466 for WD; ATP7B 589 for WD; ATP7B 621 for WD; ATP7B 887 for WD; ATP7B 1056 for WD; and ATP7B 1061 for WD. In particular embodiments, signature peptides include the peptides disclosed in FIG. 1: CD3ε 197 for SCID; WASp 274 for WAS; WASp 289 for WAS; BTK 407 for XLA; BTK 545 for XLA; CTNS 115 for cystinosis; CTNS 360 for cystinosis; SHPK 363 for cystinosis; ATP7B 325 for WD; and ATP7B 1056 for WD. In particular embodiments, signature peptides include the peptides of Table 1: CTNS 120 for cystinosis; CTNS 194 for cystinosis; SHPK 44 for cystinosis; SHPK 388 for cystinosis; ATP7B 214 for WD; ATP7B 466 for WD; ATP7B 589 for WD; ATP7B 621 for WD; ATP7B 887 for WD; and ATP7B 1061 for WD. In particular embodiments, enriching for signature peptides include contacting mixtures of peptide fragments from digested DBS with one or more binding entities that recognize the signature peptides. In particular embodiments, the binding entities are antibodies or antigen binding fragments thereof. In particular embodiments, the antibodies include those disclosed in Tables 2 and 3 above. In particular embodiments, amino acid sequences of antibodies of the disclosure include SEQ ID NOs: 79, 81, 83, 85, 86, 88, 90, 91, 93, 95, 96, 98, 100, 101, 103, 105, 106, 108, 110, 111, 113, 115, 116, 118, 120, 121, 123, 125, 126, 128, 130, 131, 133, 135, 136, 138, 140, and 141. In particular embodiments, coding sequences of antibodies of the disclosure include SEQ ID NOs: 78, 80, 82, 84, 87, 89, 92, 94, 97, 99, 102, 104, 107, 109, 112, 114, 117, 119, 122, 124, 127, 129, 132, 134, 137, and 139. In particular embodiments, the antibodies include antibodies that bind CD3ε 197, WASp 274, WASp 289, BTK 407, BTK 545, CTNS 115, CTNS 120, CTNS 194, SHPK 44, SHPK 363, SHPK 388, ATP7B 214, ATP7B 325, ATP7B 466, ATP7B 589, ATP7B 621, ATP7B 887, ATP7B 1056, and ATP7B 1061.
In particular embodiments, antibodies including SEQ ID NOs: 22-27, 63, and 64 are used to enrich for a CD3ε peptide including SEQ ID NO: 1.
In particular embodiments, antibodies including SEQ ID NOs: 28-33, 65 and 66 are used to enrich for a WASp peptide including SEQ ID NO: 2. In particular embodiments, antibodies are used to enrich for a WASp peptide including SEQ ID NO: 3.
In particular embodiments, antibodies including SEQ ID NOs: 34-38, 67, and 68 are used to enrich for a BTK peptide including SEQ ID NO: 4. In particular embodiments, antibodies are used to enrich for a BTK peptide including SEQ ID NO: 5.
In particular embodiments, the following combination of antibodies can be used to screen for SCID, WAS, and XLA: antibodies including SEQ ID NOs: 22-27, 63, and 64 that bind to a CD3ε peptide including SEQ ID NO: 1; antibodies including SEQ ID NOs: 28-33, 65 and 66 that bind to a WASp peptide including SEQ ID NO: 2; antibodies that bind to a WASp peptide including SEQ ID NO: 3; antibodies including SEQ ID NOs: 34-38, 67, and 68 that bind to a BTK peptide including SEQ ID NO: 4; and antibodies that bind to a BTK peptide including SEQ ID NO: 5.
In particular embodiments, the following combination of antibodies can be used to screen for SCID, WAS, and XLA: antibodies including SEQ ID NOs: 22-27, 63, and 64 that bind to a CD3ε peptide including SEQ ID NO: 1; antibodies including SEQ ID NOs: 28-33, 65 and 66 that bind to a WASp peptide including SEQ ID NO: 2; antibodies that bind to a WASp peptide including SEQ ID NO: 3; and antibodies including SEQ ID NOs: 34-38, 67, and 68 that bind to a BTK peptide including SEQ ID NO: 4.
In particular embodiments, the following combination of antibodies can be used to screen for SCID, WAS, and XLA: antibodies including SEQ ID NOs: 22-27, 63, and 64 that bind to a CD3ε peptide including SEQ ID NO: 1; antibodies including SEQ ID NOs: 28-33, 65 and 66 that bind to a WASp peptide including SEQ ID NO: 2; antibodies including SEQ ID NOs: 34-38, 67, and 68 that bind to a BTK peptide including SEQ ID NO: 4; and antibodies that bind to a BTK peptide including SEQ ID NO: 5.
In particular embodiments, the following combination of antibodies can be used to screen for SCID, WAS, and XLA: antibodies including SEQ ID NOs: 22-27, 63, and 64 that bind to a CD3ε peptide including SEQ ID NO: 1; antibodies including SEQ ID NOs: 28-33, 65 and 66 that bind to a WASp peptide including SEQ ID NO: 2; and antibodies including SEQ ID NOs: 34-38, 67, and 68 that bind to a BTK peptide including SEQ ID NO: 4.
In particular embodiments, antibodies including SEQ ID NOs: 39-44, 69, and 70 are used to enrich for a CTNS peptide including SEQ ID NO: 6. In particular embodiments, antibodies including SEQ ID NOs: 45-50, 71, and 72 are used to enrich for a CTNS peptide including SEQ ID NOs: 7 and 8. In particular embodiments, antibodies are used to enrich for a CTNS peptide of SEQ ID NO: 12. In particular embodiments, antibodies are used to enrich for a CTNS peptide of SEQ ID NO: 13.
In particular embodiments, antibodies including SEQ ID NOs: 51-56, 73, and 74 are used to enrich for a SHPK peptide including SEQ ID NO: 9. In particular embodiments, antibodies are used to enrich for a SHPK peptide of SEQ ID NO: 14. In particular embodiments, antibodies are used to enrich for a SHPK peptide of SEQ ID NO: 15.
In particular embodiments, the following combination of antibodies can be used to screen for cystinosis: antibodies including SEQ ID NOs: 39-44, 69, and 70 that bind to a CTNS peptide including SEQ ID NO: 6; antibodies including SEQ ID NOs: 45-50, 71, and 72 that bind to a CTNS peptide including SEQ ID NOs: 7 and 8; antibodies that bind to a CTNS peptide including SEQ ID NO: 12; antibodies that bind to a CTNS peptide including SEQ ID NO: 13; antibodies including SEQ ID NOs: 51-56, 73, and 74 that bind to a SHPK peptide including SEQ ID NO: 9; antibodies that bind to a SHPK peptide including SEQ ID NO: 14; and antibodies that bind to a SHPK peptide including SEQ ID NO: 15.
In particular embodiments, the following combination of antibodies can be used to screen for cystinosis: antibodies including SEQ ID NOs: 39-44, 69, and 70 that bind to a CTNS peptide including SEQ ID NO: 6; antibodies including SEQ ID NOs: 45-50, 71, and 72 that bind to a CTNS peptide including SEQ ID NOs: 7 and 8; antibodies that bind to a CTNS peptide including SEQ ID NO: 12; antibodies that bind to a CTNS peptide including SEQ ID NO: 13; antibodies including SEQ ID NOs: 51-56, 73, and 74 that bind to a SHPK peptide including SEQ ID NO: 9; and antibodies that bind to a SHPK peptide including SEQ ID NO: 14.
In particular embodiments, the following combination of antibodies can be used to screen for cystinosis: antibodies including SEQ ID NOs: 39-44, 69, and 70 that bind to a CTNS peptide including SEQ ID NO: 6; antibodies including SEQ ID NOs: 45-50, 71, and 72 that bind to a CTNS peptide including SEQ ID NOs: 7 and 8; antibodies that bind to a CTNS peptide including SEQ ID NO: 12; antibodies that bind to a CTNS peptide including SEQ ID NO: 13; antibodies including SEQ ID NOs: 51-56, 73, and 74 that bind to a SHPK peptide including SEQ ID NO: 9; and antibodies that bind to a SHPK peptide including SEQ ID NO: 15.
In particular embodiments, the following combination of antibodies can be used to screen for cystinosis: antibodies including SEQ ID NOs: 39-44, 69, and 70 that bind to a CTNS peptide including SEQ ID NO: 6; antibodies including SEQ ID NOs: 45-50, 71, and 72 that bind to a CTNS peptide including SEQ ID NOs: 7 and 8; antibodies that bind to a CTNS peptide including SEQ ID NO: 12; antibodies that bind to a CTNS peptide including SEQ ID NO: 13; antibodies that bind to a SHPK peptide including SEQ ID NO: 14; and antibodies that bind to a SHPK peptide including SEQ ID NO: 15.
In particular embodiments, the following combination of antibodies can be used to screen for cystinosis: antibodies including SEQ ID NOs: 39-44, 69, and 70 that bind to a CTNS peptide including SEQ ID NO: 6; antibodies including SEQ ID NOs: 45-50, 71, and 72 that bind to a CTNS peptide including SEQ ID NOs: 7 and 8; antibodies that bind to a CTNS peptide including SEQ ID NO: 12; antibodies including SEQ ID NOs: 51-56, 73, and 74 that bind to a SHPK peptide including SEQ ID NO: 9; antibodies that bind to a SHPK peptide including SEQ ID NO: 14; and antibodies that bind to a SHPK peptide including SEQ ID NO: 15.
In particular embodiments, the following combination of antibodies can be used to screen for cystinosis: antibodies including SEQ ID NOs: 39-44, 69, and 70 that bind to a CTNS peptide including SEQ ID NO: 6; antibodies including SEQ ID NOs: 45-50, 71, and 72 that bind to a CTNS peptide including SEQ ID NOs: 7 and 8; antibodies that bind to a CTNS peptide including SEQ ID NO: 13; antibodies including SEQ ID NOs: 51-56, 73, and 74 that bind to a SHPK peptide including SEQ ID NO: 9; antibodies that bind to a SHPK peptide including SEQ ID NO: 14; and antibodies that bind to a SHPK peptide including SEQ ID NO: 15.
In particular embodiments, the following combination of antibodies can be used to screen for cystinosis: antibodies including SEQ ID NOs: 39-44, 69, and 70 that bind to a CTNS peptide including SEQ ID NO: 6; antibodies that bind to a CTNS peptide including SEQ ID NO: 12; antibodies that bind to a CTNS peptide including SEQ ID NO: 13; antibodies including SEQ ID NOs: 51-56, 73, and 74 that bind to a SHPK peptide including SEQ ID NO: 9; antibodies that bind to a SHPK peptide including SEQ ID NO: 14; and antibodies that bind to a SHPK peptide including SEQ ID NO: 15.
In particular embodiments, the following combination of antibodies can be used to screen for cystinosis: antibodies including SEQ ID NOs: 45-50, 71, and 72 that bind to a CTNS peptide including SEQ ID NOs: 7 and 8; antibodies that bind to a CTNS peptide including SEQ ID NO: 12; antibodies that bind to a CTNS peptide including SEQ ID NO: 13; antibodies including SEQ ID NOs: 51-56, 73, and 74 that bind to a SHPK peptide including SEQ ID NO: 9; antibodies that bind to a SHPK peptide including SEQ ID NO: 14; and antibodies that bind to a SHPK peptide including SEQ ID NO: 15.
In particular embodiments, the following combination of antibodies can be used to screen for cystinosis: antibodies including SEQ ID NOs: 39-44, 69, and 70 that bind to a CTNS peptide including SEQ ID NO: 6; antibodies including SEQ ID NOs: 45-50, 71, and 72 that bind to a CTNS peptide including SEQ ID NOs: 7 and 8; antibodies that bind to a CTNS peptide including SEQ ID NO: 12; antibodies that bind to a CTNS peptide including SEQ ID NO: 13; and antibodies including SEQ ID NOs: 51-56, 73, and 74 that bind to a SHPK peptide including SEQ ID NO: 9.
In particular embodiments, the following combination of antibodies can be used to screen for cystinosis: antibodies including SEQ ID NOs: 39-44, 69, and 70 that bind to a CTNS peptide including SEQ ID NO: 6; antibodies including SEQ ID NOs: 45-50, 71, and 72 that bind to a CTNS peptide including SEQ ID NOs: 7 and 8; antibodies that bind to a CTNS peptide including SEQ ID NO: 12; antibodies that bind to a CTNS peptide including SEQ ID NO: 13; and antibodies that bind to a SHPK peptide including SEQ ID NO: 14.
In particular embodiments, the following combination of antibodies can be used to screen for cystinosis: antibodies including SEQ ID NOs: 39-44, 69, and 70 that bind to a CTNS peptide including SEQ ID NO: 6; antibodies including SEQ ID NOs: 45-50, 71, and 72 that bind to a CTNS peptide including SEQ ID NOs: 7 and 8; antibodies that bind to a CTNS peptide including SEQ ID NO: 12; antibodies including SEQ ID NOs: 51-56, 73, and 74 that bind to a SHPK peptide including SEQ ID NO: 9; and antibodies that bind to a SHPK peptide including SEQ ID NO: 14.
In particular embodiments, the following combination of antibodies can be used to screen for cystinosis: antibodies including SEQ ID NOs: 39-44, 69, and 70 that bind to a CTNS peptide including SEQ ID NO: 6; antibodies including SEQ ID NOs: 45-50, 71, and 72 that bind to a CTNS peptide including SEQ ID NOs: 7 and 8; antibodies that bind to a CTNS peptide including SEQ ID NO: 13; antibodies including SEQ ID NOs: 51-56, 73, and 74 that bind to a SHPK peptide including SEQ ID NO: 9; and antibodies that bind to a SHPK peptide including SEQ ID NO: 14.
In particular embodiments, the following combination of antibodies can be used to screen for cystinosis: antibodies including SEQ ID NOs: 39-44, 69, and 70 that bind to a CTNS peptide including SEQ ID NO: 6; antibodies that bind to a CTNS peptide including SEQ ID NO: 12; antibodies that bind to a CTNS peptide including SEQ ID NO: 13; antibodies including SEQ ID NOs: 51-56, 73, and 74 that bind to a SHPK peptide including SEQ ID NO: 9; and antibodies that bind to a SHPK peptide including SEQ ID NO: 14.
In particular embodiments, the following combination of antibodies can be used to screen for cystinosis: antibodies including SEQ ID NOs: 45-50, 71, and 72 that bind to a CTNS peptide including SEQ ID NOs: 7 and 8; antibodies that bind to a CTNS peptide including SEQ ID NO: 12; antibodies that bind to a CTNS peptide including SEQ ID NO: 13; antibodies including SEQ ID NOs: 51-56, 73, and 74 that bind to a SHPK peptide including SEQ ID NO: 9; and antibodies that bind to a SHPK peptide including SEQ ID NO: 14.
In particular embodiments, the following combination of antibodies can be used to screen for cystinosis: antibodies that bind to a CTNS peptide including SEQ ID NO: 12; antibodies that bind to a CTNS peptide including SEQ ID NO: 13; antibodies including SEQ ID NOs: 51-56, 73, and 74 that bind to a SHPK peptide including SEQ ID NO: 9; antibodies that bind to a SHPK peptide including SEQ ID NO: 14; and antibodies that bind to a SHPK peptide including SEQ ID NO: 15.
In particular embodiments, the following combination of antibodies can be used to screen for cystinosis: antibodies including SEQ ID NOs: 45-50, 71, and 72 that bind to a CTNS peptide including SEQ ID NOs: 7 and 8; antibodies that bind to a CTNS peptide including SEQ ID NO: 13; antibodies including SEQ ID NOs: 51-56, 73, and 74 that bind to a SHPK peptide including SEQ ID NO: 9; antibodies that bind to a SHPK peptide including SEQ ID NO: 14; and antibodies that bind to a SHPK peptide including SEQ ID NO: 15.
In particular embodiments, the following combination of antibodies can be used to screen for cystinosis: antibodies including SEQ ID NOs: 45-50, 71, and 72 that bind to a CTNS peptide including SEQ ID NOs: 7 and 8; antibodies that bind to a CTNS peptide including SEQ ID NO: 12; antibodies including SEQ ID NOs: 51-56, 73, and 74 that bind to a SHPK peptide including SEQ ID NO: 9; antibodies that bind to a SHPK peptide including SEQ ID NO: 14; and antibodies that bind to a SHPK peptide including SEQ ID NO: 15.
In particular embodiments, the following combination of antibodies can be used to screen for cystinosis: antibodies including SEQ ID NOs: 45-50, 71, and 72 that bind to a CTNS peptide including SEQ ID NOs: 7 and 8; antibodies that bind to a CTNS peptide including SEQ ID NO: 12; antibodies that bind to a CTNS peptide including SEQ ID NO: 13; antibodies that bind to a SHPK peptide including SEQ ID NO: 14; and antibodies that bind to a SHPK peptide including SEQ ID NO: 15.
In particular embodiments, the following combination of antibodies can be used to screen for cystinosis: antibodies including SEQ ID NOs: 45-50, 71, and 72 that bind to a CTNS peptide including SEQ ID NOs: 7 and 8; antibodies that bind to a CTNS peptide including SEQ ID NO: 12; antibodies that bind to a CTNS peptide including SEQ ID NO: 13; antibodies including SEQ ID NOs: 51-56, 73, and 74 that bind to a SHPK peptide including SEQ ID NO: 9; and antibodies that bind to a SHPK peptide including SEQ ID NO: 15.
In particular embodiments, the following combination of antibodies can be used to screen for cystinosis: antibodies including SEQ ID NOs: 39-44, 69, and 70 that bind to a CTNS peptide including SEQ ID NO: 6; antibodies that bind to a CTNS peptide including SEQ ID NO: 13; antibodies including SEQ ID NOs: 51-56, 73, and 74 that bind to a SHPK peptide including SEQ ID NO: 9; antibodies that bind to a SHPK peptide including SEQ ID NO: 14; and antibodies that bind to a SHPK peptide including SEQ ID NO: 15.
In particular embodiments, the following combination of antibodies can be used to screen for cystinosis: antibodies including SEQ ID NOs: 39-44, 69, and 70 that bind to a CTNS peptide including SEQ ID NO: 6; antibodies that bind to a CTNS peptide including SEQ ID NO: 12; antibodies including SEQ ID NOs: 51-56, 73, and 74 that bind to a SHPK peptide including SEQ ID NO: 9; antibodies that bind to a SHPK peptide including SEQ ID NO: 14; and antibodies that bind to a SHPK peptide including SEQ ID NO: 15.
In particular embodiments, the following combination of antibodies can be used to screen for cystinosis: antibodies including SEQ ID NOs: 39-44, 69, and 70 that bind to a CTNS peptide including SEQ ID NO: 6; antibodies that bind to a CTNS peptide including SEQ ID NO: 12; antibodies that bind to a CTNS peptide including SEQ ID NO: 13; antibodies that bind to a SHPK peptide including SEQ ID NO: 14; and antibodies that bind to a SHPK peptide including SEQ ID NO: 15.
In particular embodiments, the following combination of antibodies can be used to screen for cystinosis: antibodies including SEQ ID NOs: 39-44, 69, and 70 that bind to a CTNS peptide including SEQ ID NO: 6; antibodies that bind to a CTNS peptide including SEQ ID NO: 12; antibodies that bind to a CTNS peptide including SEQ ID NO: 13; antibodies including SEQ ID NOs: 51-56, 73, and 74 that bind to a SHPK peptide including SEQ ID NO: 9; and antibodies that bind to a SHPK peptide including SEQ ID NO: 15.
In particular embodiments, the following combination of antibodies can be used to screen for cystinosis: antibodies including SEQ ID NOs: 39-44, 69, and 70 that bind to a CTNS peptide including SEQ ID NO: 6; antibodies including SEQ ID NOs: 45-50, 71, and 72 that bind to a CTNS peptide including SEQ ID NOs: 7 and 8; antibodies including SEQ ID NOs: 51-56, 73, and 74 that bind to a SHPK peptide including SEQ ID NO: 9; antibodies that bind to a SHPK peptide including SEQ ID NO: 14; and antibodies that bind to a SHPK peptide including SEQ ID NO: 15.
In particular embodiments, the following combination of antibodies can be used to screen for cystinosis: antibodies including SEQ ID NOs: 39-44, 69, and 70 that bind to a CTNS peptide including SEQ ID NO: 6; antibodies including SEQ ID NOs: 45-50, 71, and 72 that bind to a CTNS peptide including SEQ ID NOs: 7 and 8; antibodies that bind to a CTNS peptide including SEQ ID NO: 13; antibodies that bind to a SHPK peptide including SEQ ID NO: 14; and antibodies that bind to a SHPK peptide including SEQ ID NO: 15.
In particular embodiments, the following combination of antibodies can be used to screen for cystinosis: antibodies including SEQ ID NOs: 39-44, 69, and 70 that bind to a CTNS peptide including SEQ ID NO: 6; antibodies including SEQ ID NOs: 45-50, 71, and 72 that bind to a CTNS peptide including SEQ ID NOs: 7 and 8; antibodies that bind to a CTNS peptide including SEQ ID NO: 13; antibodies including SEQ ID NOs: 51-56, 73, and 74 that bind to a SHPK peptide including SEQ ID NO: 9; and antibodies that bind to a SHPK peptide including SEQ ID NO: 15.
In particular embodiments, the following combination of antibodies can be used to screen for cystinosis: antibodies including SEQ ID NOs: 39-44, 69, and 70 that bind to a CTNS peptide including SEQ ID NO: 6; antibodies including SEQ ID NOs: 45-50, 71, and 72 that bind to a CTNS peptide including SEQ ID NOs: 7 and 8; antibodies that bind to a CTNS peptide including SEQ ID NO: 12; antibodies that bind to a SHPK peptide including SEQ ID NO: 14; and antibodies that bind to a SHPK peptide including SEQ ID NO: 15.
In particular embodiments, the following combination of antibodies can be used to screen for cystinosis: antibodies including SEQ ID NOs: 39-44, 69, and 70 that bind to a CTNS peptide including SEQ ID NO: 6; antibodies including SEQ ID NOs: 45-50, 71, and 72 that bind to a CTNS peptide including SEQ ID NOs: 7 and 8; antibodies that bind to a CTNS peptide including SEQ ID NO: 12; antibodies including SEQ ID NOs: 51-56, 73, and 74 that bind to a SHPK peptide including SEQ ID NO: 9; and antibodies that bind to a SHPK peptide including SEQ ID NO: 15.
In particular embodiments, the following combination of antibodies can be used to screen for cystinosis: antibodies including SEQ ID NOs: 39-44, 69, and 70 that bind to a CTNS peptide including SEQ ID NO: 6; antibodies including SEQ ID NOs: 45-50, 71, and 72 that bind to a CTNS peptide including SEQ ID NOs: 7 and 8; antibodies that bind to a CTNS peptide including SEQ ID NO: 12; antibodies that bind to a CTNS peptide including SEQ ID NO: 13; and antibodies that bind to a SHPK peptide including SEQ ID NO: 15.
In particular embodiments, the following combination of antibodies can be used to screen for cystinosis: antibodies including SEQ ID NOs: 39-44, 69, and 70 that bind to a CTNS peptide including SEQ ID NO: 6; antibodies including SEQ ID NOs: 45-50, 71, and 72 that bind to a CTNS peptide including SEQ ID NOs: 7 and 8; and antibodies including SEQ ID NOs: 51-56, 73, and 74 that bind to a SHPK peptide including SEQ ID NO: 9.
In particular embodiments, the following combination of antibodies can be used to screen for cystinosis: antibodies including SEQ ID NOs: 39-44, 69, and 70 that bind to a CTNS peptide including SEQ ID NO: 6; and antibodies including SEQ ID NOs: 51-56, 73, and 74 that bind to a SHPK peptide including SEQ ID NO: 9.
One of ordinary skill in the art can recognize that other combinations of antibodies selected from the following can be used to screen for cystinosis: antibodies including SEQ ID NOs: 39-44, 69, and 70 that bind to a CTNS peptide including SEQ ID NO: 6; antibodies including SEQ ID NOs: 45-50, 71, and 72 that bind to a CTNS peptide including SEQ ID NOs: 7 and 8; antibodies that bind to a CTNS peptide including SEQ ID NO: 12; antibodies that bind to a CTNS peptide including SEQ ID NO: 13; antibodies including SEQ ID NOs: 51-56, 73, and 74 that bind to a SHPK peptide including SEQ ID NO: 9; antibodies that bind to a SHPK peptide including SEQ ID NO: 14; and antibodies that bind to a SHPK peptide including SEQ ID NO: 15.
In particular embodiments, antibodies are used to enrich for an ATP7B peptide including SEQ ID NO: 10. In particular embodiments, antibodies including SEQ ID NOs: 57-62, 75, and 76 are used to enrich for an ATP7B peptide including SEQ ID NO: 11 or 21. In particular embodiments, antibodies are used to enrich for an ATP7B peptide of SEQ ID NO: 16. In particular embodiments, antibodies are used to enrich for an ATP7B peptide of SEQ ID NO: 17. In particular embodiments, antibodies are used to enrich for an ATP7B peptide of SEQ ID NO: 18. In particular embodiments, antibodies are used to enrich for an ATP7B peptide of SEQ ID NO: 19. In particular embodiments, antibodies are used to enrich for an ATP7B peptide of SEQ ID NO: 20. In particular embodiments, antibodies are used to enrich for an ATP7B peptide of SEQ ID NO: 21.
In particular embodiments, the following combination of antibodies can be used to screen for WD: antibodies that bind to an ATP7B peptide including SEQ ID NO: 10; antibodies including SEQ ID NOs: 57-62, 75, and 76 that bind to an ATP7B peptide including SEQ ID NO: 11 or 21; antibodies that bind to an ATP7B peptide including SEQ ID NO: 16; antibodies that bind to an ATP7B peptide including SEQ ID NO: 17; antibodies that bind to an ATP7B peptide including SEQ ID NO: 18; antibodies that bind to an ATP7B peptide including SEQ ID NO: 19; antibodies that bind to an ATP7B peptide including SEQ ID NO: 20; and antibodies that bind to an ATP7B peptide including SEQ ID NO: 21.
In particular embodiments, the following combination of antibodies can be used to screen for WD: antibodies that bind to an ATP7B peptide including SEQ ID NO: 10; antibodies including SEQ ID NOs: 57-62, 75, and 76 that bind to an ATP7B peptide including SEQ ID NO: 11 or 21; antibodies that bind to an ATP7B peptide including SEQ ID NO: 16; antibodies that bind to an ATP7B peptide including SEQ ID NO: 17; antibodies that bind to an ATP7B peptide including SEQ ID NO: 18; antibodies that bind to an ATP7B peptide including SEQ ID NO: 19; and antibodies that bind to an ATP7B peptide including SEQ ID NO: 20.
In particular embodiments, the following combination of antibodies can be used to screen for WD: antibodies that bind to an ATP7B peptide including SEQ ID NO: 10; antibodies including SEQ ID NOs: 57-62, 75, and 76 that bind to an ATP7B peptide including SEQ ID NO: 11 or 21; antibodies that bind to an ATP7B peptide including SEQ ID NO: 16; antibodies that bind to an ATP7B peptide including SEQ ID NO: 17; antibodies that bind to an ATP7B peptide including SEQ ID NO: 18; antibodies that bind to an ATP7B peptide including SEQ ID NO: 19; and antibodies that bind to an ATP7B peptide including SEQ ID NO: 21.
In particular embodiments, the following combination of antibodies can be used to screen for WD: antibodies that bind to an ATP7B peptide including SEQ ID NO: 10; antibodies including SEQ ID NOs: 57-62, 75, and 76 that bind to an ATP7B peptide including SEQ ID NO: 11 or 21; antibodies that bind to an ATP7B peptide including SEQ ID NO: 16; antibodies that bind to an ATP7B peptide including SEQ ID NO: 17; antibodies that bind to an ATP7B peptide including SEQ ID NO: 18; antibodies that bind to an ATP7B peptide including SEQ ID NO: 20; and antibodies that bind to an ATP7B peptide including SEQ ID NO: 21.
In particular embodiments, the following combination of antibodies can be used to screen for WD: antibodies that bind to an ATP7B peptide including SEQ ID NO: 10; antibodies including SEQ ID NOs: 57-62, 75, and 76 that bind to an ATP7B peptide including SEQ ID NO: 11 or 21; antibodies that bind to an ATP7B peptide including SEQ ID NO: 16; antibodies that bind to an ATP7B peptide including SEQ ID NO: 17; antibodies that bind to an ATP7B peptide including SEQ ID NO: 19; antibodies that bind to an ATP7B peptide including SEQ ID NO: 20; and antibodies that bind to an ATP7B peptide including SEQ ID NO: 21.
In particular embodiments, the following combination of antibodies can be used to screen for WD: antibodies that bind to an ATP7B peptide including SEQ ID NO: 10; antibodies including SEQ ID NOs: 57-62, 75, and 76 that bind to an ATP7B peptide including SEQ ID NO: 11 or 21; antibodies that bind to an ATP7B peptide including SEQ ID NO: 16; antibodies that bind to an ATP7B peptide including SEQ ID NO: 18; antibodies that bind to an ATP7B peptide including SEQ ID NO: 19; antibodies that bind to an ATP7B peptide including SEQ ID NO: 20; and antibodies that bind to an ATP7B peptide including SEQ ID NO: 21.
In particular embodiments, the following combination of antibodies can be used to screen for WD: antibodies that bind to an ATP7B peptide including SEQ ID NO: 10; antibodies including SEQ ID NOs: 57-62, 75, and 76 that bind to an ATP7B peptide including SEQ ID NO: 11 or 21; antibodies that bind to an ATP7B peptide including SEQ ID NO: 17; antibodies that bind to an ATP7B peptide including SEQ ID NO: 18; antibodies that bind to an ATP7B peptide including SEQ ID NO: 19; antibodies that bind to an ATP7B peptide including SEQ ID NO: 20; and antibodies that bind to an ATP7B peptide including SEQ ID NO: 21.
In particular embodiments, the following combination of antibodies can be used to screen for WD: antibodies that bind to an ATP7B peptide including SEQ ID NO: 10; antibodies that bind to an ATP7B peptide including SEQ ID NO: 16; antibodies that bind to an ATP7B peptide including SEQ ID NO: 17; antibodies that bind to an ATP7B peptide including SEQ ID NO: 18; antibodies that bind to an ATP7B peptide including SEQ ID NO: 19; antibodies that bind to an ATP7B peptide including SEQ ID NO: 20; and antibodies that bind to an ATP7B peptide including SEQ ID NO:
In particular embodiments, the following combination of antibodies can be used to screen for WD: antibodies including SEQ ID NOs: 57-62, 75, and 76 that bind to an ATP7B peptide including SEQ ID NO: 11 or 21; antibodies that bind to an ATP7B peptide including SEQ ID NO: 16; antibodies that bind to an ATP7B peptide including SEQ ID NO: 17; antibodies that bind to an ATP7B peptide including SEQ ID NO: 18; antibodies that bind to an ATP7B peptide including SEQ ID NO: 19; antibodies that bind to an ATP7B peptide including SEQ ID NO: 20; and antibodies that bind to an ATP7B peptide including SEQ ID NO: 21.
In particular embodiments, the following combination of antibodies can be used to screen for WD: antibodies that bind to an ATP7B peptide including SEQ ID NO: 10; antibodies including SEQ ID NOs: 57-62, 75, and 76 that bind to an ATP7B peptide including SEQ ID NO: 11 or 21; antibodies that bind to an ATP7B peptide including SEQ ID NO: 16; antibodies that bind to an ATP7B peptide including SEQ ID NO: 17; antibodies that bind to an ATP7B peptide including SEQ ID NO: 18; and antibodies that bind to an ATP7B peptide including SEQ ID NO: 19.
In particular embodiments, the following combination of antibodies can be used to screen for WD: antibodies that bind to an ATP7B peptide including SEQ ID NO: 10; antibodies including SEQ ID NOs: 57-62, 75, and 76 that bind to an ATP7B peptide including SEQ ID NO: 11 or 21; antibodies that bind to an ATP7B peptide including SEQ ID NO: 16; antibodies that bind to an ATP7B peptide including SEQ ID NO: 17; antibodies that bind to an ATP7B peptide including SEQ ID NO: 18; and antibodies that bind to an ATP7B peptide including SEQ ID NO: 20.
In particular embodiments, the following combination of antibodies can be used to screen for WD: antibodies that bind to an ATP7B peptide including SEQ ID NO: 10; antibodies including SEQ ID NOs: 57-62, 75, and 76 that bind to an ATP7B peptide including SEQ ID NO: 11 or 21; antibodies that bind to an ATP7B peptide including SEQ ID NO: 16; antibodies that bind to an ATP7B peptide including SEQ ID NO: 17; antibodies that bind to an ATP7B peptide including SEQ ID NO: 19; and antibodies that bind to an ATP7B peptide including SEQ ID NO: 20,
In particular embodiments, the following combination of antibodies can be used to screen for WD: antibodies that bind to an ATP7B peptide including SEQ ID NO: 10; antibodies including SEQ ID NOs: 57-62, 75, and 76 that bind to an ATP7B peptide including SEQ ID NO: 11 or 21; antibodies that bind to an ATP7B peptide including SEQ ID NO: 16; antibodies that bind to an ATP7B peptide including SEQ ID NO: 18; antibodies that bind to an ATP7B peptide including SEQ ID NO: 19; and antibodies that bind to an ATP7B peptide including SEQ ID NO: 20.
In particular embodiments, the following combination of antibodies can be used to screen for WD: antibodies that bind to an ATP7B peptide including SEQ ID NO: 10; antibodies including SEQ ID NOs: 57-62, 75, and 76 that bind to an ATP7B peptide including SEQ ID NO: 11 or 21; antibodies that bind to an ATP7B peptide including SEQ ID NO: 17; antibodies that bind to an ATP7B peptide including SEQ ID NO: 18; antibodies that bind to an ATP7B peptide including SEQ ID NO: 19; and antibodies that bind to an ATP7B peptide including SEQ ID NO: 20.
In particular embodiments, the following combination of antibodies can be used to screen for WD: antibodies that bind to an ATP7B peptide including SEQ ID NO: 10; antibodies that bind to an ATP7B peptide including SEQ ID NO: 16; antibodies that bind to an ATP7B peptide including SEQ ID NO: 17; antibodies that bind to an ATP7B peptide including SEQ ID NO: 18; antibodies that bind to an ATP7B peptide including SEQ ID NO: 19; and antibodies that bind to an ATP7B peptide including SEQ ID NO: 20.
In particular embodiments, the following combination of antibodies can be used to screen for WD: antibodies including SEQ ID NOs: 57-62, 75, and 76 that bind to an ATP7B peptide including SEQ ID NO: 11 or 21; antibodies that bind to an ATP7B peptide including SEQ ID NO: 16; antibodies that bind to an ATP7B peptide including SEQ ID NO: 17; antibodies that bind to an ATP7B peptide including SEQ ID NO: 18; antibodies that bind to an ATP7B peptide including SEQ ID NO: 19; and antibodies that bind to an ATP7B peptide including SEQ ID NO: 20.
In particular embodiments, the following combination of antibodies can be used to screen for WD: antibodies that bind to an ATP7B peptide including SEQ ID NO: 16; antibodies that bind to an ATP7B peptide including SEQ ID NO: 17; antibodies that bind to an ATP7B peptide including SEQ ID NO: 18; antibodies that bind to an ATP7B peptide including SEQ ID NO: 19; antibodies that bind to an ATP7B peptide including SEQ ID NO: 20; and antibodies that bind to an ATP7B peptide including SEQ ID NO: 21.
In particular embodiments, the following combination of antibodies can be used to screen for WD: antibodies including SEQ ID NOs: 57-62, 75, and 76 that bind to an ATP7B peptide including SEQ ID NO: 11 or 21; antibodies that bind to an ATP7B peptide including SEQ ID NO: 17; antibodies that bind to an ATP7B peptide including SEQ ID NO: 18; antibodies that bind to an ATP7B peptide including SEQ ID NO: 19; antibodies that bind to an ATP7B peptide including SEQ ID NO: 20; and antibodies that bind to an ATP7B peptide including SEQ ID NO: 21.
In particular embodiments, the following combination of antibodies can be used to screen for WD: antibodies including SEQ ID NOs: 57-62, 75, and 76 that bind to an ATP7B peptide including SEQ ID NO: 11 or 21; antibodies that bind to an ATP7B peptide including SEQ ID NO: 16; antibodies that bind to an ATP7B peptide including SEQ ID NO: 18; antibodies that bind to an ATP7B peptide including SEQ ID NO: 19; antibodies that bind to an ATP7B peptide including SEQ ID NO: 20; and antibodies that bind to an ATP7B peptide including SEQ ID NO: 21.
In particular embodiments, the following combination of antibodies can be used to screen for WD: antibodies including SEQ ID NOs: 57-62, 75, and 76 that bind to an ATP7B peptide including SEQ ID NO: 11 or 21; antibodies that bind to an ATP7B peptide including SEQ ID NO: 16; antibodies that bind to an ATP7B peptide including SEQ ID NO: 17; antibodies that bind to an ATP7B peptide including SEQ ID NO: 19; antibodies that bind to an ATP7B peptide including SEQ ID NO: 20; and antibodies that bind to an ATP7B peptide including SEQ ID NO: 21.
In particular embodiments, the following combination of antibodies can be used to screen for WD: antibodies including SEQ ID NOs: 57-62, 75, and 76 that bind to an ATP7B peptide including SEQ ID NO: 11 or 21; antibodies that bind to an ATP7B peptide including SEQ ID NO: 16; antibodies that bind to an ATP7B peptide including SEQ ID NO: 17; antibodies that bind to an ATP7B peptide including SEQ ID NO: 18; antibodies that bind to an ATP7B peptide including SEQ ID NO: 20; and antibodies that bind to an ATP7B peptide including SEQ ID NO: 21.
In particular embodiments, the following combination of antibodies can be used to screen for WD: antibodies including SEQ ID NOs: 57-62, 75, and 76 that bind to an ATP7B peptide including SEQ ID NO: 11 or 21; antibodies that bind to an ATP7B peptide including SEQ ID NO: 16; antibodies that bind to an ATP7B peptide including SEQ ID NO: 17; antibodies that bind to an ATP7B peptide including SEQ ID NO: 18; antibodies that bind to an ATP7B peptide including SEQ ID NO: 19; and antibodies that bind to an ATP7B peptide including SEQ ID NO: 21.
In particular embodiments, the following combination of antibodies can be used to screen for WD: antibodies that bind to an ATP7B peptide including SEQ ID NO: 10; antibodies including SEQ ID NOs: 57-62, 75, and 76 that bind to an ATP7B peptide including SEQ ID NO: 11 or 21; antibodies that bind to an ATP7B peptide including SEQ ID NO: 16; antibodies that bind to an ATP7B peptide including SEQ ID NO: 19; antibodies that bind to an ATP7B peptide including SEQ ID NO: 20; and antibodies that bind to an ATP7B peptide including SEQ ID NO: 21.
In particular embodiments, the following combination of antibodies can be used to screen for WD: antibodies that bind to an ATP7B peptide including SEQ ID NO: 10; antibodies including SEQ ID NOs: 57-62, 75, and 76 that bind to an ATP7B peptide including SEQ ID NO: 11 or 21; antibodies that bind to an ATP7B peptide including SEQ ID NO: 16; antibodies that bind to an ATP7B peptide including SEQ ID NO: 18; antibodies that bind to an ATP7B peptide including SEQ ID NO: 20; and antibodies that bind to an ATP7B peptide including SEQ ID NO: 21.
In particular embodiments, the following combination of antibodies can be used to screen for WD: antibodies that bind to an ATP7B peptide including SEQ ID NO: 10; antibodies including SEQ ID NOs: 57-62, 75, and 76 that bind to an ATP7B peptide including SEQ ID NO: 11 or 21; antibodies that bind to an ATP7B peptide including SEQ ID NO: 16; antibodies that bind to an ATP7B peptide including SEQ ID NO: 18; antibodies that bind to an ATP7B peptide including SEQ ID NO: 19; and antibodies that bind to an ATP7B peptide including SEQ ID NO: 21.
In particular embodiments, the following combination of antibodies can be used to screen for WD: antibodies that bind to an ATP7B peptide including SEQ ID NO: 10; antibodies including SEQ ID NOs: 57-62, 75, and 76 that bind to an ATP7B peptide including SEQ ID NO: 11 or 21; antibodies that bind to an ATP7B peptide including SEQ ID NO: 16; antibodies that bind to an ATP7B peptide including SEQ ID NO: 17; antibodies that bind to an ATP7B peptide including SEQ ID NO: 20; and antibodies that bind to an ATP7B peptide including SEQ ID NO: 21.
In particular embodiments, the following combination of antibodies can be used to screen for WD: antibodies that bind to an ATP7B peptide including SEQ ID NO: 10; antibodies including SEQ ID NOs: 57-62, 75, and 76 that bind to an ATP7B peptide including SEQ ID NO: 11 or 21; antibodies that bind to an ATP7B peptide including SEQ ID NO: 16; antibodies that bind to an ATP7B peptide including SEQ ID NO: 17; antibodies that bind to an ATP7B peptide including SEQ ID NO: 19; and antibodies that bind to an ATP7B peptide including SEQ ID NO: 21.
In particular embodiments, the following combination of antibodies can be used to screen for WD: antibodies that bind to an ATP7B peptide including SEQ ID NO: 10; antibodies including SEQ ID NOs: 57-62, 75, and 76 that bind to an ATP7B peptide including SEQ ID NO: 11 or 21; antibodies that bind to an ATP7B peptide including SEQ ID NO: 16; antibodies that bind to an ATP7B peptide including SEQ ID NO: 17; antibodies that bind to an ATP7B peptide including SEQ ID NO: 18; and antibodies that bind to an ATP7B peptide including SEQ ID NO: 21.
In particular embodiments, the following combination of antibodies can be used to screen for WD: antibodies including SEQ ID NOs: 57-62, 75, and 76 that bind to an ATP7B peptide including SEQ ID NO: 11 or 21; antibodies that bind to an ATP7B peptide including SEQ ID NO: 16; and antibodies that bind to an ATP7B peptide including SEQ ID NO: 20.
One of ordinary skill in the art can recognize that other combinations of antibodies selected from the following can be used to screen for WD: antibodies that bind to an ATP7B peptide including SEQ ID NO: 10; antibodies including SEQ ID NOs: 57-62, 75, and 76 that bind to an ATP7B peptide including SEQ ID NO: 11 or 21; antibodies that bind to an ATP7B peptide including SEQ ID NO: 16; antibodies that bind to an ATP7B peptide including SEQ ID NO: 17; antibodies that bind to an ATP7B peptide including SEQ ID NO: 18; antibodies that bind to an ATP7B peptide including SEQ ID NO: 19; antibodies that bind to an ATP7B peptide including SEQ ID NO: 20; and antibodies that bind to an ATP7B peptide including SEQ ID NO: 21.
The methods of the present disclosure include optionally performing liquid chromatography on the immunoaffinity enriched peptides to separate the peptides prior to MS analysis. Liquid chromatography can separate peptides based on their weight and affinity for the mobile and stationary phases of the column.
The methods of the present disclosure include performing SRM-MS or MRM-MS on the immunoaffinity enriched peptides to quantify the amount of a given signature peptide. In particular embodiments, the SRM-MS or MRM-MS is carried out as described above. In particular embodiments, the quantification of a signature peptide includes using a reference peptide that is introduced into an assay in known amounts. In particular embodiments, a reference peptide can be identical to the signature peptide in every respect except that the reference peptide has been differentially labeled, for example, with one or more heavy isotopes, to distinguish the reference peptide from the signature peptide.
In particular embodiments, SRM-MS or MRM-MS detects a reduction or absence in a CD3ε peptide. In particular embodiments, the CD3ε peptide includes SEQ ID NO: 1.
In particular embodiments, SRM-MS or MRM-MS detects a reduction or absence in a WASp peptide. In particular embodiments, the WASp peptide includes SEQ ID NO: 2. In particular embodiments, the WASp peptide includes SEQ ID NO: 3.
In particular embodiments, SRM-MS or MRM-MS detects a reduction or absence in a BTK peptide. In particular embodiments, the BTK peptide includes SEQ ID NO: 4. In particular embodiments, the BTK peptide includes SEQ ID NO: 5.
In particular embodiments, SRM-MS or MRM-MS detects a reduction or absence in a CTNS peptide. In particular embodiments, the CTNS peptide includes SEQ ID NO: 6. In particular embodiments, the CTNS peptide includes SEQ ID NOs: 7 and 8. In particular embodiments, the CTNS peptide includes SEQ ID NO: 12. In particular embodiments, the CTNS peptide includes SEQ ID NO: 13.
In particular embodiments, SRM-MS or MRM-MS detects a reduction or absence in a SHPK peptide. In particular embodiments, the SHPK peptide includes SEQ ID NO: 9. In particular embodiments, the SHPK peptide includes SEQ ID NO: 14. In particular embodiments, the SHPK peptide includes SEQ ID NO: 15.
In particular embodiments, SRM-MS or MRM-MS detects a reduction or absence in an ATP7B peptide. In particular embodiments, the ATP7B peptide includes SEQ ID NO: 10. In particular embodiments, the ATP7B peptide includes SEQ ID NO: 11 or 21. In particular embodiments, the ATP7B peptide includes SEQ ID NO: 16. In particular embodiments, the ATP7B peptide includes SEQ ID NO: 17. In particular embodiments, the ATP7B peptide includes SEQ ID NO: 18. In particular embodiments, the ATP7B peptide includes SEQ ID NO: 19. In particular embodiments, the ATP7B peptide includes SEQ ID NO: 20. In particular embodiments, the ATP7B peptide includes SEQ ID NO: 21.
In particular embodiments, a predetermined cut-off value is used as a threshold for a given signature peptide. A concentration of a given signature peptide above the threshold indicates that the assayed DBS is from an individual not afflicted by SCID, WAS, XLA, cystinosis, or WD. A concentration of a given signature peptide below the threshold or absent indicates that the assayed DBS is from an individual afflicted by SCID, WAS, XLA, cystinosis, or WD. In particular embodiments, the threshold can be determined by analysis of a population of normal controls and calculation of standard deviation (SD) of a concentration of a given signature peptide in this population. The threshold can be set at a certain SD from the mean concentration of the given signature peptide. In particular embodiments, the threshold is −1 SD, −1.1 SD, −1.2 SD, −1.3 SD, −1.4 SD, −1.5 SD, −1.6 SD, −1.7 SD, −1.8 SD, −1.9 SD, −2.0 SD, −2.1 SD, −2.2 SD, −2.3 SD, −2.4 SD, −2.5 SD, −2.6 SD, −2.7 SD, −2.8 SD, −2.9 SD, −3.0 SD, or more SD from the mean concentration of the given signature peptide. In particular embodiments, for diagnosis or screening of SCID, WAS, XLA, or cystinosis, the threshold can be determined by analysis of a population of normal controls and calculation of standard deviation (SD) of a ratio of a concentration of a given signature peptide to an endogenous concentration of ATP7B in this population. Peptide concentration cutoffs for each PIDD can be set at a certain SD derived from mean concentration of each signature peptide or ratio of a concentration of a given signature peptide to an endogenous concentration of ATP7B.
In particular embodiments, the threshold concentration for the CD3ε 197 peptide includes −1.0 SD, −1.25 SD, −1.3 SD, −1.35 SD, −1.4 SD, −1.45 SD, −1.5 SD, −1.55 SD, −1.6 SD, −1.65 SD, −1.7 SD, −1.75 SD, −1.8 SD, −1.85 SD, −1.9 SD, −1.95 SD, −2.0 SD, −2.25 SD, −2.3 SD, −2.35 SD, −2.4 SD, −2.45 SD, −2.5 SD, −2.55 SD, −2.6 SD, −2.65 SD, −2.7 SD, −2.75 SD, −2.8 SD, −2.85 SD, −2.9 SD, −2.95 SD, −3.0 SD, or more from the mean concentration of CD3ε 197 in a population of normal controls. In particular embodiments, the threshold concentration for the CD3ε 197 peptide includes 60 pmol/L or less, 55 pmol/L or less, 50 pmol/L or less, 45 pmol/L or less, 40 pmol/L or less, 39 pmol/L or less, 38 pmol/L or less, 37 pmol/L or less, 36 pmol/L or less, 35 pmol/L or less, 34 pmol/L or less, 33 pmol/L or less, 32 pmol/L or less, 31 pmol/L or less, 30 pmol/L or less.
In particular embodiments, the threshold concentration for the WASp 274 peptide includes −1.0 SD, −1.25 SD, −1.3 SD, −1.35 SD, −1.4 SD, −1.45 SD, −1.5 SD, −1.55 SD, −1.6 SD, −1.65 SD, −1.7 SD, −1.75 SD, −1.8 SD, −1.85 SD, −1.9 SD, −1.95 SD, −2.0 SD, −2.25 SD, −2.3 SD, −2.35 SD, −2.4 SD, −2.45 SD, −2.5 SD, −2.55 SD, −2.6 SD, −2.65 SD, −2.7 SD, −2.75 SD, −2.8 SD, −2.85 SD, −2.9 SD, −2.95 SD, −3.0 SD, or more from the mean concentration of WASp 274 in a population of normal controls. In particular embodiments, the threshold concentration for the WASp 274 peptide includes 220 pmol/L or less, 215 pmol/L or less, 210 pmol/L or less, 205 pmol/L or less, 200 pmol/L or less, 195 pmol/L or less, 190 pmol/L or less, 185 pmol/L or less, 180 pmol/L or less, 175 pmol/L or less, 170 pmol/L or less, 165 pmol/L or less, 160 pmol/L or less, 155 pmol/L or less, 150 pmol/L or less.
In particular embodiments, the threshold concentration for the WASp 289 peptide includes −1.0 SD, −1.25 SD, −1.3 SD, −1.35 SD, −1.4 SD, −1.45 SD, −1.5 SD, −1.55 SD, −1.6 SD, −1.65 SD, −1.7 SD, −1.75 SD, −1.8 SD, −1.85 SD, −1.9 SD, −1.95 SD, −2.0 SD, −2.25 SD, −2.3 SD, −2.35 SD, −2.4 SD, −2.45 SD, −2.5 SD, −2.55 SD, −2.6 SD, −2.65 SD, −2.7 SD, −2.75 SD, −2.8 SD, −2.85 SD, −2.9 SD, −2.95 SD, −3.0 SD, or more from the mean concentration of WASp 289 in a population of normal controls. In particular embodiments, the threshold concentration for the WASp 289 peptide includes 2500 pmol/L or less, 2490 pmol/L or less, 2480 pmol/L or less, 2470 pmol/L or less, 2460 pmol/L or less, 2450 pmol/L or less, 2440 pmol/L or less, 2430 pmol/L or less, 2420 pmol/L or less, 2410 pmol/L or less, 2400 pmol/L or less.
In particular embodiments, the threshold concentration for the BTK 407 peptide includes −1.0 SD, −1.25 SD, −1.3 SD, −1.35 SD, −1.4 SD, −1.45 SD, −1.5 SD, −1.55 SD, −1.6 SD, −1.65 SD, −1.7 SD, −1.75 SD, −1.8 SD, −1.85 SD, −1.9 SD, −1.95 SD, −2.0 SD, −2.25 SD, −2.3 SD, −2.35 SD, −2.4 SD, −2.45 SD, −2.5 SD, −2.55 SD, −2.6 SD, −2.65 SD, −2.7 SD, −2.75 SD, −2.8 SD, −2.85 SD, −2.9 SD, −2.95 SD, −3.0 SD, or more from the mean concentration of BTK 407 in a population of normal controls. In particular embodiments, the threshold concentration for the BTK 407 peptide includes 80 pmol/L or less, 75 pmol/L or less, 70 pmol/L or less, 65 pmol/L or less, 60 pmol/L or less, 55 pmol/L or less, 50 pmol/L or less, 49 pmol/L or less, 48 pmol/L or less, 47 pmol/L or less, 46 pmol/L or less, 45 pmol/L or less, 44 pmol/L or less, 43 pmol/L or less, 42 pmol/L or less, 41 pmol/L or less, 40 pmol/L or less, 35 pmol/L or less, 30 pmol/L or less.
In particular embodiments, the threshold concentration for the BTK 545 peptide includes −1.0 SD, −1.25 SD, −1.3 SD, −1.35 SD, −1.4 SD, −1.45 SD, −1.5 SD, −1.55 SD, −1.6 SD, −1.65 SD, −1.7 SD, −1.75 SD, −1.8 SD, −1.85 SD, −1.9 SD, −1.95 SD, −2.0 SD, −2.25 SD, −2.3 SD, −2.35 SD, −2.4 SD, −2.45 SD, −2.5 SD, −2.55 SD, −2.6 SD, −2.65 SD, −2.7 SD, −2.75 SD, −2.8 SD, −2.85 SD, −2.9 SD, −2.95 SD, −3.0 SD, or more from the mean concentration of BTK 545 in a population of normal controls. In particular embodiments, the threshold concentration for the BTK 545 peptide includes 110 pmol/L or less, 109 pmol/L or less, 108 pmol/L or less, 107 pmol/L or less, 106 pmol/L or less, 105 pmol/L or less, 104 pmol/L or less, 103 pmol/L or less, 102 pmol/L or less, 101 pmol/L or less, 100 pmol/L or less.
In particular embodiments, the threshold concentration for the CTNS 115 peptide includes −1.0 SD, −1.25 SD, −1.3 SD, −1.35 SD, −1.4 SD, −1.45 SD, −1.5 SD, −1.55 SD, −1.6 SD, −1.65 SD, −1.7 SD, −1.75 SD, −1.8 SD, −1.85 SD, −1.9 SD, −1.95 SD, −2.0 SD, −2.25 SD, −2.3 SD, −2.35 SD, −2.4 SD, −2.45 SD, −2.5 SD, −2.55 SD, −2.6 SD, −2.65 SD, −2.7 SD, −2.75 SD, −2.8 SD, −2.85 SD, −2.9 SD, −2.95 SD, −3.0 SD, or more from the mean concentration of CTNS 115 in a population of normal controls. In particular embodiments, the threshold concentration for the CTNS 115 peptide includes 60 pmol/L or less, 59 pmol/L or less, 58 pmol/L or less, 57 pmol/L or less, 56 pmol/L or less, 55 pmol/L or less, 54 pmol/L or less, 53 pmol/L or less, 52 pmol/L or less, 51 pmol/L or less, 50 pmol/L or less.
In particular embodiments, the threshold concentration for the SHPK 363 peptide includes −1.0 SD, −1.25 SD, −1.3 SD, −1.35 SD, −1.4 SD, −1.45 SD, −1.5 SD, −1.55 SD, −1.6 SD, −1.65 SD, −1.7 SD, −1.75 SD, −1.8 SD, −1.85 SD, −1.9 SD, −1.95 SD, −2.0 SD, −2.25 SD, −2.3 SD, −2.35 SD, −2.4 SD, −2.45 SD, −2.5 SD, −2.55 SD, −2.6 SD, −2.65 SD, −2.7 SD, −2.75 SD, −2.8 SD, −2.85 SD, −2.9 SD, −2.95 SD, −3.0 SD, or more from the mean concentration of SHPK 363 in a population of normal controls. In particular embodiments, the threshold concentration for the SHPK 363 peptide includes 2000 pmol/L or less, 1950 pmol/L or less, 1900 pmol/L or less, 1850 pmol/L or less, 1800 pmol/L or less, 1750 pmol/L or less, 1700 pmol/L or less, 1650 pmol/L or less, 1600 pmol/L or less, 1550 pmol/L or less, 1500 pmol/L or less.
In particular embodiments, the threshold concentration for the ATP7B 1056 peptide includes −1.0 SD, −1.25 SD, −1.3 SD, −1.35 SD, −1.4 SD, −1.45 SD, −1.5 SD, −1.55 SD, −1.6 SD, −1.65 SD, −1.7 SD, −1.75 SD, −1.8 SD, −1.85 SD, −1.9 SD, −1.95 SD, −2.0 SD, −2.25 SD, −2.3 SD, −2.35 SD, −2.4 SD, −2.45 SD, −2.5 SD, −2.55 SD, −2.6 SD, −2.65 SD, −2.7 SD, −2.75 SD, −2.8 SD, −2.85 SD, −2.9 SD, −2.95 SD, −3.0 SD, or more from the mean concentration of ATP7B 1056 in a population of normal controls. In particular embodiments, the threshold concentration for the ATP7B 1056 peptide includes 90 pmol/L or less, 85 pmol/L or less, 80 pmol/L or less, 75 pmol/L or less, 70 pmol/L or less, 65 pmol/L or less, 60 pmol/L or less, 55 pmol/L or less, 50 pmol/L or less, 45 pmol/L or less, 40 pmol/L or less, 35 pmol/L or less, 30 pmol/L or less.
In particular embodiments, the threshold concentration for the ATP7B 214 peptide includes −1.0 SD, −1.25 SD, −1.3 SD, −1.35 SD, −1.4 SD, −1.45 SD, −1.5 SD, −1.55 SD, −1.6 SD, −1.65 SD, −1.7 SD, −1.75 SD, −1.8 SD, −1.85 SD, −1.9 SD, −1.95 SD, −2.0 SD, −2.25 SD, −2.3 SD, −2.35 SD, −2.4 SD, −2.45 SD, −2.5 SD, −2.55 SD, −2.6 SD, −2.65 SD, −2.7 SD, −2.75 SD, −2.8 SD, −2.85 SD, −2.9 SD, −2.95 SD, −3.0 SD, or more from the mean concentration of ATP7B 214 in a population of normal controls. In particular embodiments, the threshold concentration for the ATP7B 214 peptide includes 30 pmol/L or less, 29 pmol/L or less, 28 pmol/L or less, 27 pmol/L or less, 26 pmol/L or less, 25 pmol/L or less, 24 pmol/L or less, 23 pmol/L or less, 22 pmol/L or less, 21 pmol/L or less, 20 pmol/L or less.
In particular embodiments, the threshold concentration for the ATP7B 887 peptide includes −1.0 SD, −1.25 SD, −1.3 SD, −1.35 SD, −1.4 SD, −1.45 SD, −1.5 SD, −1.55 SD, −1.6 SD, −1.65 SD, −1.7 SD, −1.75 SD, −1.8 SD, −1.85 SD, −1.9 SD, −1.95 SD, −2.0 SD, −2.25 SD, −2.3 SD, −2.35 SD, −2.4 SD, −2.45 SD, −2.5 SD, −2.55 SD, −2.6 SD, −2.65 SD, −2.7 SD, −2.75 SD, −2.8 SD, −2.85 SD, −2.9 SD, −2.95 SD, −3.0 SD, or more from the mean concentration of ATP7B 887 in a population of normal controls. In particular embodiments, the threshold concentration for the ATP7B 887 peptide includes 190 pmol/L or less, 185 pmol/L or less, 180 pmol/L or less, 175 pmol/L or less, 170 pmol/L or less, 165 pmol/L or less, 160 pmol/L or less, 155 pmol/L or less, 150 pmol/L or less, 145 pmol/L or less, 140 pmol/L or less.
In particular embodiments, a signature peptide can be considered a primary biomarker for diagnosis or screening of a given disease. A primary signature peptide can include peptides that are used first to diagnose or screen for a given disease. In particular embodiments, a primary marker can be reproducibly obtained from a digestion of the corresponding protein, has high affinity antibodies for immunoaffinity enrichment, and/or is reproducible across independent liquid chromatography columns and/or mass spectrometry instruments. In particular embodiments, a signature peptide can be considered a secondary marker for diagnosis or screening of a given disease. A secondary signature peptide can include peptides that are used second to confirm a diagnosis or screening of a given disease with a primary marker. In particular embodiments, BTK 545 can be a secondary marker to BTK 407 in diagnosing XLA. In particular embodiments, WASp 289 can be a secondary marker to WASp 274 in diagnosing WAS.
In particular embodiments, antibodies of the present disclosure can also be used in complimentary clinical tests for the diagnosis of primary immunodeficiencies, cystinosis, and WD for those patients with ambiguous biochemical results, and for patients who carry the variants of unknown significance from genetic tests.
Methods disclosed herein include treating subjects (e.g., humans) based upon the outcome of screening for SCID, WAS, XLA, cystinosis, and/or WD with compositions and methods disclosed herein. Treating subjects includes delivering therapeutically effective amounts. Therapeutically effective amounts include those that provide effective amounts, prophylactic treatments and/or therapeutic treatments.
An “effective amount” is the amount of a composition necessary to result in a desired physiological change in the subject. For example, an effective amount can provide an alleviation of symptoms, an elimination of symptoms, or a cure for SCID, WAS, XLA, cystinosis, and/or WD. Effective amounts are often administered for research purposes. Effective amounts disclosed herein can cause a statistically-significant effect in an animal model or in vitro assay relevant to the assessment of a disease's development, progression, and/or resolution.
Particular embodiments may include administering compositions as a “prophylactic treatment.” Prophylactic treatments include those administered to a subject who does not display signs or symptoms of SCID, WAS, XLA, cystinosis, and/or WD or displays only early signs or symptoms of SCID, WAS, XLA, cystinosis, and/or WD, such that treatment is administered for the purpose of diminishing or decreasing the risk of developing the disorder. Thus, a prophylactic treatment functions as a preventative treatment against SCID, WAS, XLA, cystinosis, and/or WD.
In particular embodiments, a prophylactic treatment can prevent, delay, or reduce the onset of SCID, WAS, XLA, cystinosis, and/or WD. In particular embodiments, a prophylactic treatment can be given prior, concurrently, or after other preventative measures, such as the use of antibiotics for WAS and XLA. In particular embodiments, a prophylactic treatment can prevent or reduce the severity of symptoms or complications associated with SCID, WAS, XLA, cystinosis, and/or WD. Symptoms and complications for SCID can include: poor growth; rashes that look like eczema; chronic diarrhea; recurrent thrush in the mouth; and pneumocystis pneumonia. Symptoms and complications for WAS can include: bleeding; eczema; bloody diarrhea; and recurrent infections. Symptoms and complications for XLA can include: infections; diarrhea; failure to grow; joint disease; kidney inflammation; red blood cell breakdown; and skin and muscle inflammation. Symptoms and complications for cystinosis can include: polyuria; polydipsia; dehydration; vomiting; metabolic acidosis; hypophosphatemic rickets; constipation; failure to thrive; recurrent bouts of fever; heat intolerance; and poor/loss of appetite. Symptoms and complications for WD can include: fatigue; lack of appetite or abdominal pain; jaundice; golden-brown eye discoloration (Kayser-Fleischer rings); fluid buildup in the legs or abdomen; problems with speech, swallowing or physical coordination; and uncontrolled movements or muscle stiffness.
A “therapeutic treatment” includes a treatment administered to a subject who displays symptoms or signs of SCID, WAS, XLA, cystinosis, and/or WD and is administered to the subject for the purpose of diminishing or eliminating those signs or symptoms of SCID, WAS, XLA, cystinosis, and/or WD. In particular embodiments, the therapeutic treatment can provide immune function for subjects diagnosed with SCID, WAS, and/or XLA. In particular embodiments, the therapeutic treatment can reduce cystine accumulation in the cells of subjects afflicted with cystinosis. In particular embodiments, the therapeutic treatment can reduce copper accumulation in organs of subjects afflicted with WD. In particular embodiments, the therapeutic treatment can reduce, control, or eliminate symptoms and complications of SCID, WAS, XLA, cystinosis, and/or WD such as those described above.
Prophylactic treatments and therapeutic treatments need not be mutually exclusive, and in particular embodiments, administered dosages may accomplish more than one treatment type.
In particular embodiments, therapeutically effective amounts provide immune system function for subjects diagnosed with SCID, WAS, and/or XLA. Thus, in particular embodiments, methods of treatment disclosed herein include stem cell transplants, immunoglobulin infusions, antibiotic infusions, and/or gene therapy, for disorders such as SCID, WAS, and XLA. In particular embodiments, methods of treatment include enzyme therapy for SCID. Providing immune function include: decreasing the frequency or number of bacterial, viral, or parasitic infections, increasing life expectancy, and/or increasing growth.
In particular embodiments, therapeutically effective amounts prevent accumulation of cystine in cells of subjects diagnosed with cystinosis. In particular embodiments, methods of treatment include providing cysteamine for cystinosis. In particular embodiments, providing cysteamine alleviates or eliminates symptoms of cystinosis as described above.
In particular embodiments, therapeutically effective amounts prevent accumulation of copper in organs of subjects diagnosed with WD. In particular embodiments, methods of treatment include D-penicillinamine, trientine, zinc salts, and/or liver transplants for WD. In particular embodiments, preventing accumulation of copper in organs alleviates or eliminates symptoms of WD as described above.
In particular embodiments, administration of a therapeutic composition can be accompanied with administration of a separate adjuvant. Exemplary adjuvants include alum, bentonite, latex, and acrylic particles; incomplete Freund's adjuvant, complete Freund's adjuvant; aluminum-based salts such as aluminum hydroxide; calcium-based salts; silica or any TLR biological ligand(s); Sigma Adjuvant System (SAS); Ribi adjuvants.
For administration, therapeutically effective amounts (also referred to herein as doses) can be initially estimated based on results from in vitro assays and/or animal model studies. Such information can be used to more accurately determine useful doses in subjects of interest. The actual dose amount administered to a particular subject can be determined by a physician, veterinarian or researcher taking into account parameters such as physical and physiological factors including target, body weight, severity of condition, previous or concurrent therapeutic interventions, idiopathy of the subject and route of administration.
Therapeutically effective amounts of cells can range from 10⁴cells/kg to 10⁹cells/kg. In particular embodiments, a therapeutically effective amount of cells can include 10⁴cells/kg, 10⁵cells/kg, 10⁶cells/kg, 10⁷cells/kg, 10⁸cells/kg, 10⁹cells/kg, or more.
Useful doses can range from 0.1 to 5 μg/kg or from 0.5 to 1 μg/kg. In particular embodiments, a dose can include 1 μg/kg, 15 μg/kg, 30 μg/kg, 50 μg/kg, 55 μg/kg, 70 μg/kg, 90 μg/kg, 150 μg/kg, 350 μg/kg, 500 μg/kg, 750 μg/kg, 1000 μg/kg, 0.1 to 5 mg/kg or from 0.5 to 1 mg/kg. In particular embodiments, a dose can include 1 mg/kg, 10 mg/kg, 30 mg/kg, 50 mg/kg, 70 mg/kg, 100 mg/kg, 300 mg/kg, 500 mg/kg, 700 mg/kg, 1000 mg/kg or more.
Therapeutically effective amounts can be achieved by administering single or multiple doses during the course of a treatment regimen (e.g., daily, every other day, every 3 days, every 4 days, every 5 days, every 6 days, weekly, every 2 weeks, every 3 weeks, monthly, every 2 months, every 3 months, every 4 months, every 5 months, every 6 months, every 7 months, every 8 months, every 9 months, every 10 months, every 11 months or yearly).

(IX) KITS

Kits to test for congenital disorders are also provided. Kits can include lancets to prick for blood, filter cards to collect blood drops, solutions to solubilize DBS, and appropriate buffers and enzymes to digest marker proteins in the DBS. Kits can further include one or more containers including anti-peptide binding agents (e.g., antibodies) and/or reagents or supplies to assess absence or reduction in CD3ε, WASp, BTK, CTNS, SHPK and/or ATP7B. In particular embodiments, the kits include one or more containers including the following anti-peptide antibodies: anti-CD3ε 197, anti-WASp 274, anti-WASp 289, anti-BTK 407, anti-BTK 545, anti-CTNS 115, anti-CTNS 120, anti-CTNS 194, anti-CTNS 360, anti-SHPK 44, anti-SHPK 363, anti-SHPK 388, anti-ATP7B 214, anti-ATP7B 325, anti-ATP7B 466, anti-ATP7B 589, anti-ATP7B 621, anti-ATP7B 887, anti-ATP7B 1056, and/or anti-ATP7B 1061. The antibodies may be immobilized on a solid support, such as a column or beads. Kits can further include elution buffers to release peptides from antibodies. In particular embodiments, kits can include one or more labeled reference peptides to perform absolute quantification of the signature peptides. In particular embodiments, kits can also include some or all of the necessary laboratory and/or medical supplies needed to use the kit effectively, such as gauze, sterile adhesive strips, gloves, tubes, and the like. Variations in contents of any of the kits described herein can be made.
Components of the kit can be prepared for storage and later use. Associated with such container(s) can be a notice in the form prescribed by a governmental agency regulating the manufacture, use, or sale of the kit, which notice reflects approval by the agency of manufacture, use, or sale, when required.
Optionally, the kits further include instructions for using the kit in the methods. In various embodiments, the instructions can include appropriate instructions to interpret results associated with using the kit; proper disposal of the related waste; and the like. The instructions can be in the form of printed instructions provided within the kit or the instructions can be printed on a portion of the kit itself. Instructions may be in the form of a sheet, pamphlet, brochure, CD-ROM, or computer-readable device, or can provide directions to instructions at a remote location, such as a website.
Variants of the sequences disclosed and referenced herein are also included. Guidance in determining which amino acid residues can be substituted, inserted, or deleted without abolishing biological activity can be found using computer programs well known in the art, such as DNASTAR™ (Madison, Wis.) software. Preferably, amino acid changes in the protein variants disclosed herein are conservative amino acid changes, i.e., substitutions of similarly charged or uncharged amino acids. A conservative amino acid change involves substitution of one of a family of amino acids which are related in their side chains.
In a peptide or protein, suitable conservative substitutions of amino acids are known to those of skill in this art and generally can be made without altering a biological activity of a resulting molecule. Those of skill in this art recognize that, in general, single amino acid substitutions in non-essential regions of a polypeptide do not substantially alter biological activity (see, e.g., Watson et al. Molecular Biology of the Gene, 4th Edition, 1987, The Benjamin/Cummings Pub. Co., p. 224). Naturally occurring amino acids are generally divided into conservative substitution families as follows: Group 1: Alanine (Ala), Glycine (Gly), Serine (Ser), and Threonine (Thr); Group 2: (acidic): Aspartic acid (Asp), and Glutamic acid (Glu); Group 3: (acidic; also classified as polar, negatively charged residues and their amides): Asparagine (Asn), Glutamine (Gin), Asp, and Glu; Group 4: Gln and Asn; Group 5: (basic; also classified as polar, positively charged residues): Arginine (Arg), Lysine (Lys), and Histidine (His); Group 6 (large aliphatic, nonpolar residues): Isoleucine (Ile), Leucine (Leu), Methionine (Met), Valine (Val) and Cysteine (Cys); Group 7 (uncharged polar): Tyrosine (Tyr), Gly, Asn, Gln, Cys, Ser, and Thr; Group 8 (large aromatic residues): Phenylalanine (Phe), Tryptophan (Trp), and Tyr; Group 9 (nonpolar): Proline (Pro), Ala, Val, Leu, Ile, Phe, Met, and Trp; Group 11 (aliphatic): Gly, Ala, Val, Leu, and Ile; Group 10 (small aliphatic, nonpolar or slightly polar residues): Ala, Ser, Thr, Pro, and Gly; and Group 12 (sulfur-containing): Met and Cys. Additional information can be found in Creighton (1984) Proteins, W.H. Freeman and Company.
In making such changes, the hydropathic index of amino acids may be considered. The importance of the hydropathic amino acid index in conferring interactive biologic function on a protein is generally understood in the art (Kyte and Doolittle, 1982, J. Mol. Biol. 157(1), 105-32). Each amino acid has been assigned a hydropathic index on the basis of its hydrophobicity and charge characteristics (Kyte and Doolittle, 1982). These values are: Ile (+4.5); Val (+4.2); Leu (+3.8); Phe (+2.8); Cys (+2.5); Met (+1.9); Ala (+1.8); Gly (−0.4); Thr (−0.7); Ser (−0.8); Trp (−0.9); Tyr (−1.3); Pro (−1.6); His (−3.2); Glutamate (−3.5); Gln (−3.5); aspartate (−3.5); Asn (−3.5); Lys (−3.9); and Arg (−4.5).
It is known in the art that certain amino acids may be substituted by other amino acids having a similar hydropathic index or score and still result in a protein with similar biological activity, i.e., still obtain a biological functionally equivalent protein. In making such changes, the substitution of amino acids whose hydropathic indices are within ±2 is preferred, those within ±1 are particularly preferred, and those within ±0.5 are even more particularly preferred. It is also understood in the art that the substitution of like amino acids can be made effectively on the basis of hydrophilicity.
As detailed in U.S. Pat. No. 4,554,101, the following hydrophilicity values have been assigned to amino acid residues: Arg (+3.0); Lys (+3.0); aspartate (+3.0±1); glutamate (+3.0±1); Ser (+0.3); Asn (+0.2); Gln (+0.2); Gly (0); Thr (−0.4); Pro (−0.5±1); Ala (−0.5); His (−0.5); Cys (−1.0); Met (−1.3); Val (−1.5); Leu (−1.8); Ile (−1.8); Tyr (−2.3); Phe (−2.5); Trp (−3.4). It is understood that an amino acid can be substituted for another having a similar hydrophilicity value and still obtain a biologically equivalent, and in particular, an immunologically equivalent protein. In such changes, the substitution of amino acids whose hydrophilicity values are within ±2 is preferred, those within ±1 are particularly preferred, and those within ±0.5 are even more particularly preferred.
As outlined above, amino acid substitutions may be based on the relative similarity of the amino acid side-chain substituents, for example, their hydrophobicity, hydrophilicity, charge, size, and the like.
As indicated elsewhere, variants of gene sequences can include codon optimized variants, sequence polymorphisms, splice variants, and/or mutations that do not affect the function of an encoded product to a statistically-significant degree.
Variants of the protein, nucleic acid, and gene sequences disclosed herein also include sequences with at least 70% sequence identity, 80% sequence identity, 85% sequence, 90% sequence identity, 95% sequence identity, 96% sequence identity, 97% sequence identity, 98% sequence identity, or 99% sequence identity to the protein, nucleic acid, or gene sequences disclosed herein.
“% sequence identity” refers to a relationship between two or more sequences, as determined by comparing the sequences. In the art, “identity” also means the degree of sequence relatedness between protein, nucleic acid, or gene sequences as determined by the match between strings of such sequences. “Identity” (often referred to as “similarity”) can be readily calculated by known methods, including (but not limited to) those described in: Computational Molecular Biology (Lesk, A. M., ed.) Oxford University Press, N Y (1988); Biocomputing: Informatics and Genome Projects (Smith, D. W., ed.) Academic Press, N Y (1994); Computer Analysis of Sequence Data, Part I (Griffin, A. M., and Griffin, H. G., eds.) Humana Press, N J (1994); Sequence Analysis in Molecular Biology (Von Heijne, G., ed.) Academic Press (1987); and Sequence Analysis Primer (Gribskov, M. and Devereux, J., eds.) Oxford University Press, NY (1992). Preferred methods to determine identity are designed to give the best match between the sequences tested. Methods to determine identity and similarity are codified in publicly available computer programs. Sequence alignments and percent identity calculations may be performed using the Megalign program of the LASERGENE bioinformatics computing suite (DNASTAR, Inc., Madison, Wis.). Multiple alignment of the sequences can also be performed using the Clustal method of alignment (Higgins and Sharp CABIOS, 5, 151-153 (1989) with default parameters (GAP PENALTY=10, GAP LENGTH PENALTY=10). Relevant programs also include the GCG suite of programs (Wisconsin Package Version 9.0, Genetics Computer Group (GCG), Madison, Wis.); BLASTP, BLASTN, BLASTX (Altschul, et al., J. Mol. Biol. 215:403-410 (1990); DNASTAR (DNASTAR, Inc., Madison, Wis.); and the FASTA program incorporating the Smith-Waterman algorithm (Pearson, Comput. Methods Genome Res., [Proc. Int. Symp.] (1994), Meeting Date 1992, 111-20. Editor(s): Suhai, Sandor. Publisher: Plenum, New York, N.Y. Within the context of this disclosure it will be understood that where sequence analysis software is used for analysis, the results of the analysis are based on the “default values” of the program referenced. As used herein “default values” will mean any set of values or parameters, which originally load with the software when first initialized.
Variants also include nucleic acid molecules that hybridizes under stringent hybridization conditions to a sequence disclosed herein and provide the same function as the reference sequence. Exemplary stringent hybridization conditions include an overnight incubation at 42° C. in a solution including 50% formamide, 5×SSC (750 mM NaCl, 75 mM trisodium citrate), 50 mM sodium phosphate (pH 7.6), 5×Denhardt's solution, 10% dextran sulfate, and 20 μg/ml denatured, sheared salmon sperm DNA, followed by washing the filters in 0.1×SSC at 50° C. Changes in the stringency of hybridization and signal detection are primarily accomplished through the manipulation of formamide concentration (lower percentages of formamide result in lowered stringency); salt conditions, or temperature. For example, moderately high stringency conditions include an overnight incubation at 37° C. in a solution including 6×SSPE (20×SSPE=3M NaCl; 0.2M NaH2PO4; 0.02M EDTA, pH 7.4), 0.5% SDS, 30% formamide, 100 μg/ml salmon sperm blocking DNA; followed by washes at 50° C. with 1×SSPE, 0.1% SDS. In addition, to achieve even lower stringency, washes performed following stringent hybridization can be done at higher salt concentrations (e.g. 5×SSC). Variations in the above conditions may be accomplished through the inclusion and/or substitution of alternate blocking reagents used to suppress background in hybridization experiments. Typical blocking reagents include Denhardt's reagent, BLOTTO, heparin, denatured salmon sperm DNA, and commercially available proprietary formulations. The inclusion of specific blocking reagents may require modification of the hybridization conditions described above, due to problems with compatibility.

(X) EXEMPLARY EMBODIMENTS

1. A method of screening for severe combined immunodeficiency (SCID), Wiskott-Aldrich syndrome (WAS), and/or X-linked agammaglobulinemia (XLA) in a subject, the method including:
Obtaining a dried blood spot (DBS) sample derived from the subject;
Digesting proteins from blood of the DBS with an enzyme to yield one or more peptides;

Enriching for:

a CD3ε signature peptide of SCID with an antibody or antigen-binding fragment thereof that binds to the CD3ε signature peptide;
a first WASp signature peptide of WAS with an antibody or antigen-binding fragment thereof that binds to the first WASp signature peptide;
a second WASp signature peptide of WAS with an antibody or antigen-binding fragment thereof that binds to the second WASp signature peptide;
a first BTK signature peptide of XLA with an antibody or antigen-binding fragment thereof that binds to the first BTK signature peptide; and/or
a second BTK signature peptide with an antibody or antigen-binding fragment thereof that binds to the second BTK signature peptide;
Performing liquid chromatography-multiple reaction monitoring mass spectrometry (LC-MRM-MS) on the enriched peptides to determine concentrations of the peptides; and Diagnosing the subject with:
SCID when the concentration of the CD3ε signature peptide is lower than a predetermined threshold concentration or when the CD3ε signature peptide is absent;
WAS when the concentrations of the first and/or second WASp signature peptides are lower than corresponding predetermined threshold concentrations or when the first and/or second WASp signature peptides are absent; and/or
XLA when the concentrations of the first and/or second BTK signature peptides are lower than corresponding predetermined threshold concentrations or when the first and/or second BTK signature peptides are absent.
2. A method of embodiment 1, wherein
the CD3ε signature peptide of SCID is encoded by an amino acid sequence set forth in SEQ ID NO: 1;
the first WASp signature peptide of WAS is encoded by an amino acid sequence set forth in SEQ ID NO: 2;
the second WASp signature peptide of WAS is encoded by an amino acid sequence set forth in SEQ ID NO: 3;
the first BTK signature peptide of XLA is encoded by an amino acid sequence set forth in SEQ ID NO: 4; and/or
the second BTK signature peptide of XLA is encoded by an amino acid sequence set forth in SEQ ID NO: 5.
3. A method of embodiment 1 or 2, wherein
the antibody or antigen-binding fragment thereof that binds to the CD3ε signature peptide includes: a heavy chain variable (VH) domain including CDR1 of SEQ ID NO: 22, CDR2 of SEQ ID NO: 23, and CDR3 of SEQ ID NO: 24, and a light chain variable (VL) domain including: CDR1 of SEQ ID NO: 25, CDR2 of SEQ ID NO: 26, and CDR3 of SEQ ID NO: 27;
the antibody or antigen-binding fragment thereof that binds to the first WASp signature peptide includes: a heavy chain variable (VH) domain including CDR1 of SEQ ID NO: 28, CDR2 of SEQ ID NO: 29, and CDR3 of SEQ ID NO: 30, and a light chain variable (VL) domain including: CDR1 of SEQ ID NO: 31, CDR2 of SEQ ID NO: 32, and CDR3 of SEQ ID NO: 33; and/or
the antibody or antigen-binding fragment thereof that binds to the first BTK signature peptide includes: a heavy chain variable (VH) domain including CDR1 of SEQ ID NO: 34, CDR2 of SEQ ID NO: 35, and CDR3 of sequence GDI, and a light chain variable (VL) domain including: CDR1 of SEQ ID NO: 36, CDR2 of SEQ ID NO: 37, and CDR3 of SEQ ID NO: 38.
4. A method of any one of embodiments 1-3, wherein the method is performed as part of a newborn screening (NBS) that additionally screens the subject for one or more of phenylketonuria, primary congenital hypothyroidism, cystic fibrosis, and sickle cell disease.
5. A method of any one of embodiments 1-4, wherein the method is performed in the absence of clinical symptoms of SCID, WAS, and/or XLA in the subject.
6. A method of any one of embodiments 1-5, wherein the enzyme is trypsin.
7. A method of any one of embodiments 1-6, wherein the corresponding predetermined threshold concentration for each signature peptide is calculated from a standard deviation of the mean concentration of each signature peptides in DBS from a population of normal control subjects.
8. A method of any one of embodiments 1-7, wherein the corresponding predetermined threshold concentration is −1 standard deviation (SD), −1.1 SD, −1.2 SD, −1.3 SD, −1.4 SD, −1.5 SD, −1.6 SD, −1.7 SD, −1.8 SD, −1.9 SD, −2.0 SD, −2.1 SD, −2.2 SD, −2.3 SD, −2.4 SD, −2.5 SD, −2.6 SD, −2.7 SD, −2.8 SD, −2.9 SD, −3.0 SD, or more SD from the mean concentration of each signature peptide in DBS from a population of normal control subjects.
9. A method of any one of embodiments 1-8, wherein the antibody or antigen-binding fragment thereof used for enrichment of the CD3ε signature peptide of SCID includes a VH domain of SEQ ID NO: 63.
10. A method of any one of embodiments 1-9, wherein the antibody or antigen-binding fragment thereof used for enrichment of the CD3ε signature peptide of SCID includes a VL domain of SEQ ID NO: 64.
11. A method of any one of embodiments 1-10, wherein the antibody or antigen-binding fragment thereof used for enrichment of the first WASp signature peptide of WAS includes a VH domain of SEQ ID NO: 65.
12. A method of any one of embodiments 1-11, wherein the antibody or antigen-binding fragment thereof used for enrichment of the first WASp signature peptide of WAS includes a VL domain of SEQ ID NO: 66.
13. A method of any one of embodiments 1-12, wherein the antibody or antigen-binding fragment thereof used for enrichment of the first WASp signature peptide of WAS includes a heavy chain of SEQ ID NO: 86.
14. A method of any one of embodiments 1-13, wherein the antibody or antigen-binding fragment thereof used for enrichment of the first WASp signature peptide of WAS includes a light chain of SEQ ID NO: 91.
15. A method of any one of embodiments 1-14, wherein the antibody or antigen-binding fragment thereof used for enrichment of the first BTK signature peptide of XLA includes a VH domain of SEQ ID NO: 67.
16. A method of any one of embodiments 1-15, wherein the antibody or antigen-binding fragment thereof used for enrichment of the first BTK signature peptide of XLA includes a VL domain of SEQ ID NO: 68.
17. A method of any one of embodiments 1-16, wherein the antibody or antigen-binding fragment thereof used for enrichment of the first BTK signature peptide of XLA includes a heavy chain of SEQ ID NO: 96.
18. A method of any one of embodiments 1-17, wherein the antibody or antigen-binding fragment thereof used for enrichment of the first BTK signature peptide of XLA includes a light chain of SEQ ID NO: 101.
19. A method of any one of embodiments 1-18, wherein the method further includes screening for cystinosis by enriching for:
a first CTNS signature peptide of cystinosis with an antibody or antigen-binding fragment thereof that binds to the first CTNS signature peptide;
a second CTNS signature peptide of cystinosis with an antibody or antigen-binding fragment thereof that binds to the second CTNS signature peptide;
a third CTNS signature peptide of cystinosis with an antibody or antigen-binding fragment thereof that binds to the third CTNS signature peptide;
a fourth CTNS signature peptide of cystinosis with an antibody or antigen-binding fragment thereof that binds to the fourth CTNS signature peptide;
a first SHPK signature peptide of cystinosis with an antibody or antigen-binding fragment thereof that binds to the first SHPK signature peptide;
a second SHPK signature peptide of cystinosis with an antibody or antigen-binding fragment thereof that binds to the second SHPK signature peptide; and/or a third SHPK signature peptide of cystinosis with an antibody or antigen-binding fragment thereof that binds to the third SHPK signature peptide;
and
Diagnosing the subject with cystinosis when the concentrations of the first CTNS, the second CTNS, the third CTNS, the fourth CTNS, the first SHPK, the second SHPK, and/or the third SHPK signature peptides are lower than corresponding predetermined threshold concentrations or when the first CTNS, the second CTNS, the third CTNS, the fourth CTNS, the first SHPK, the second SHPK, and/or the third SHPK signature peptides are absent.
20. A method of embodiment 19, wherein
the first CTNS signature peptide of cystinosis is encoded by an amino acid sequence set forth in SEQ ID NO: 6;
the second CTNS signature peptide of cystinosis is encoded by an amino acid sequence set forth in SEQ ID NO: 7 or 8;
the third CTNS signature peptide of cystinosis is encoded by an amino acid sequence set forth in SEQ ID NO: 12;
the fourth CTNS signature peptide of cystinosis is encoded by an amino acid sequence set forth in SEQ ID NO: 13;
the first SHPK signature peptide of cystinosis is encoded by an amino acid sequence set forth in SEQ ID NO: 9;
the second SHPK signature peptide of cystinosis is encoded by an amino acid sequence set forth in SEQ ID NO: 14; and/or
the third SHPK signature peptide of cystinosis is encoded by an amino acid sequence set forth in SEQ ID NO: 15.
21. A method of embodiment 19 or 20, wherein
the antibody or antigen-binding fragment thereof that binds to the first CTNS signature peptide of cystinosis includes: a heavy chain variable (VH) domain including CDR1 of SEQ ID NO: 39, CDR2 of SEQ ID NO: 40, and CDR3 of SEQ ID NO: 41, and a light chain variable (VL) domain including: CDR1 of SEQ ID NO: 42, CDR2 of SEQ ID NO: 43, and CDR3 of SEQ ID NO: 44;
the antibody or antigen-binding fragment thereof that binds to the second CTNS signature peptide includes: a heavy chain variable (VH) domain including CDR1 of SEQ ID NO: 45, CDR2 of SEQ ID NO: 46, and CDR3 of SEQ ID NO: 47, and a light chain variable (VL) domain including: CDR1 of SEQ ID NO: 48, CDR2 of SEQ ID NO: 49, and CDR3 of SEQ ID NO: 50; and/or
the antibody or antigen-binding fragment thereof that binds to the first SHPK signature peptide includes: a heavy chain variable (VH) domain including CDR1 of SEQ ID NO: 51, CDR2 of SEQ ID NO: 52, and CDR3 of SEQ ID NO: 53, and a light chain variable (VL) domain including: CDR1 of SEQ ID NO: 54, CDR2 of SEQ ID NO: 55, and CDR3 of SEQ ID NO: 56.
22. A method of any one of embodiments 19-21, wherein the antibody or antigen-binding fragment thereof used for enrichment of the first CTNS signature peptide of cystinosis includes a VH domain of SEQ ID NO: 69.
23. A method of any one of embodiments 19-22, wherein the antibody or antigen-binding fragment thereof used for enrichment of the first CTNS signature peptide of cystinosis includes a VL domain of SEQ ID NO: 70.
24. A method of any one of embodiments 19-23, wherein the antibody or antigen-binding fragment thereof used for enrichment of the first CTNS signature peptide of cystinosis includes a heavy chain of SEQ ID NO: 106.
25. A method of any one of embodiments 19-24, wherein the antibody or antigen-binding fragment thereof used for enrichment of the first CTNS signature peptide of cystinosis includes a light chain of SEQ ID NO: 111.
26. A method of any one of embodiments 19-25, wherein the antibody or antigen-binding fragment thereof used for enrichment of the second CTNS signature peptide of cystinosis includes a VH domain of SEQ ID NO: 71.
27. A method of any one of embodiments 19-26, wherein the antibody or antigen-binding fragment thereof used for enrichment of the second CTNS signature peptide of cystinosis includes a VL domain of SEQ ID NO: 72.
28. A method of any one of embodiments 19-27, wherein the antibody or antigen-binding fragment thereof used for enrichment of the second CTNS signature peptide of cystinosis includes a heavy chain of SEQ ID NO: 116.
29. A method of any one of embodiments 19-28, wherein the antibody or antigen-binding fragment thereof used for enrichment of the second CTNS signature peptide of cystinosis includes a light chain of SEQ ID NO: 121.
30. A method of any one of embodiments 19-29, wherein the antibody or antigen-binding fragment thereof used for enrichment of the first SHPK signature peptide of cystinosis includes a VH domain of SEQ ID NO: 73.
31. A method of any one of embodiments 19-30, wherein the antibody or antigen-binding fragment thereof used for enrichment of the first SHPK signature peptide of cystinosis includes a VL domain of SEQ ID NO: 74.
32. A method of any one of embodiments 19-31, wherein the antibody or antigen-binding fragment thereof used for enrichment of the first SHPK signature peptide of cystinosis includes a heavy chain of SEQ ID NO: 126.
33. A method of any one of embodiments 19-32, wherein the antibody or antigen-binding fragment thereof used for enrichment of the first SHPK signature peptide of cystinosis includes a light chain of SEQ ID NO: 131.
34. A method of any one of embodiments 1-33, wherein the method further includes screening for Wilson Disease (WD) by enriching for:
a first ATP7B signature peptide with an antibody or antigen-binding fragment thereof that binds to the first ATP7B signature peptide;
a second ATP7B signature peptide with an antibody or antigen-binding fragment thereof that binds to the second ATP7B signature peptide;
a third ATP7B signature peptide with an antibody or antigen-binding fragment that binds to the third ATP7B signature peptide;
a fourth ATP7B signature peptide with an antibody or antigen-binding fragment thereof that binds to the fourth ATP7B signature peptide;
a fifth ATP7B signature peptide with an antibody or antigen-binding fragment thereof that binds to the fifth ATP7B signature peptide;
a sixth ATP7B signature peptide with an antibody or antigen-binding fragment thereof that binds to the sixth ATP7B signature peptide; and/or
a seventh ATP7B signature peptide with an antibody or antigen-binding fragment thereof;
and
Diagnosing the subject with WD when the concentrations of the first ATP7B, the second ATP7B, the third ATP7B, the fourth ATP7B, the fifth ATP7B, the sixth ATP7B, and/or the seventh ATP7B signature peptides are lower than corresponding predetermined threshold concentrations or when the first ATP7B, the second ATP7B, the third ATP7B, the fourth ATP7B, the fifth ATP7B, the sixth ATP7B, and/or the seventh ATP7B signature peptides are absent.
35. A method of embodiment 34, wherein
the first ATP7B signature peptide of WD is encoded by an amino acid sequence set forth in SEQ ID NO: 10;
the second ATP7B signature peptide of WD is encoded by an amino acid sequence set forth in SEQ ID NO: 11 or 21;
the third ATP7B signature peptide of WD is encoded by an amino acid sequence set forth in SEQ ID NO: 16;
the fourth ATP7B signature peptide of WD is encoded by an amino acid sequence set forth in SEQ ID NO: 17;
the fifth ATP7B signature peptide of WD is encoded by an amino acid sequence set forth in SEQ ID NO: 18;
the sixth ATP7B signature peptide of WD is encoded by an amino acid sequence set forth in SEQ ID NO: 19; and/or
the seventh ATP7B signature peptide of WD is encoded by an amino acid sequence set forth in SEQ ID NO: 20.
36. A method of embodiment 34 or 35, wherein the antibody or antigen-binding fragment thereof that binds to the second ATP7B signature peptide includes: a heavy chain variable (VH) domain including CDR1 of SEQ ID NO: 57, CDR2 of SEQ ID NO: 58, and CDR3 of SEQ ID NO: 59, and a light chain variable (VL) domain including: CDR1 of SEQ ID NO: 60, CDR2 of SEQ ID NO: 61, and CDR3 of SEQ ID NO: 62.
37. A method of any one of embodiments 34-36, wherein the antibody or antigen-binding fragment thereof used for enrichment of the second ATP7B signature peptide of WD includes a VH domain of SEQ ID NO: 75.
38. A method of any one of embodiments 34-37, wherein the antibody or antigen-binding fragment thereof used for enrichment of the second ATP7B signature peptide of WD includes a VL domain of SEQ ID NO: 76.
39. A method of any one of embodiments 34-38, wherein the antibody or antigen-binding fragment thereof used for enrichment of the second ATP7B signature peptide of WD includes a heavy chain of SEQ ID NO: 136.
40. A method of any one of embodiments 34-39, wherein the antibody or antigen-binding fragment thereof used for enrichment of the second ATP7B signature peptide of WD includes a light chain of SEQ ID NO: 141.
41. A method of any one of embodiments 1-40, wherein the concentrations of the peptides are determined from corresponding known concentrations of reference signature peptides added prior to the LC-MRM-MS.
42. A method of any one of embodiments 7-41, wherein the mean concentration of the CD3ε signature peptide in DBS from a population of normal control subjects includes a concentration in a range of 70 pmol/L to 400 pmol/L.
43. A method of any one of embodiments 7-42, wherein the mean concentration of the first WASp signature peptide in DBS from a population of normal control subjects includes a concentration in a range of 600 pmol/L to 5000 pmol/L.
44. A method of any one of embodiments 7-43, wherein the mean concentration of the second WASp signature peptide in DBS from a population of normal control subjects includes a concentration in a range of 5500 pmol/L to 15000 pmol/L.
45. A method of any one of embodiments 7-44, wherein the mean concentration of the first BTK signature peptide in DBS from a population of normal control subjects includes a concentration in a range of 350 pmol/L to 2500 pmol/L.
46. A method of any one of embodiments 7-45, wherein the mean concentration of the second BTK signature peptide in DBS from a population of normal control subjects includes a concentration in a range of 550 pmol/L to 1600 pmol/L.
47. A method of any one of embodiments 7-46, wherein the mean concentration of the first CTNS signature peptide in DBS from a population of normal control subjects includes a concentration in a range of 40 pmol/L to 250 pmol/L.
48. A method of any one of embodiments 7-47, wherein the mean concentration of the first SHPK signature peptide in DBS from a population of normal control subjects includes a concentration in a range of 100 pmol/L to 8000 pmol/L.
49. A method of any one of embodiments 7-48, wherein the mean concentration of the second ATP7B signature peptide in DBS from a population of normal control subjects includes a concentration in a range of 90 pmol/L to 400 pmol/L.
50. A method of any one of embodiments 7-49, wherein the mean concentration of the third ATP7B signature peptide in DBS from a population of normal control subjects includes a concentration in a range of 30 pmol/L to 100 pmol/L.
51. A method of any one of embodiments 7-50, wherein the mean concentration of the seventh ATP7B signature peptide in DBS from a population of normal control subjects includes a concentration in a range of 200 pmol/L to 500 pmol/L.
52. A method of screening for severe combined immunodeficiency (SCID), Wiskott-Aldrich syndrome (WAS), and/or X-linked agammaglobulinemia (XLA) in a subject, the method including:
Obtaining a dried blood spot (DBS) sample derived from the subject;
Digesting proteins from blood of the DBS with an enzyme to yield one or more peptides;

Enriching for:

a CD3ε signature peptide of SCID with an antibody or antigen-binding fragment thereof that binds to the CD3ε signature peptide;
a first WASp signature peptide of WAS with an antibody or antigen-binding fragment thereof that binds to the first WASp signature peptide;
a second WASp signature peptide of WAS with an antibody or antigen-binding fragment thereof that binds to the second WASp signature peptide;
a first BTK signature peptide of XLA with an antibody or antigen-binding fragment thereof that binds to the first BTK signature peptide; and/or
a second BTK signature peptide with an antibody or antigen-binding fragment thereof that binds to the second BTK signature peptide;
Enriching for an endogenous ATP7B peptide with an antibody or antigen binding fragment thereof that binds to the endogenous ATP7B peptide;
Performing liquid chromatography-multiple reaction monitoring mass spectrometry (LC-MRM-MS) on the enriched peptides to determine concentrations of the peptides;
Calculating a ratio of signature peptide concentration:endogenous ATP7B peptide concentration for each signature peptide; and
Diagnosing the subject with:
SCID when the ratio of CD3ε signature peptide concentration:endogenous ATP7B peptide concentration is lower than a corresponding predetermined threshold ratio or when the CD3ε signature peptide is absent;
WAS when the ratios of the first and/or second WASp signature peptide concentration:endogenous ATP7B peptide concentration are lower than corresponding predetermined threshold ratios or when the first and/or second WASp signature peptides are absent; and/or
XLA when the ratios of the first and/or second BTK signature peptide concentration:endogenous ATP7B peptide concentration are lower than corresponding predetermined threshold ratios or when the first and/or second BTK signature peptides are absent.
53. A method of embodiment 52, wherein
the CD3ε signature peptide of SCID is encoded by an amino acid sequence set forth in SEQ ID NO: 1;
the first WASp signature peptide of WAS is encoded by an amino acid sequence set forth in SEQ ID NO: 2;
the second WASp signature peptide of WAS is encoded by an amino acid sequence set forth in SEQ ID NO: 3;
the first BTK signature peptide of XLA is encoded by an amino acid sequence set forth in SEQ ID NO: 4; and/or
the second BTK signature peptide of XLA is encoded by an amino acid sequence set forth in SEQ ID NO: 5.
54. A method of embodiment 52 or 53, wherein
the antibody or antigen-binding fragment thereof that binds to the CD3ε signature peptide includes: a heavy chain variable (VH) domain including CDR1 of SEQ ID NO: 22, CDR2 of SEQ ID NO: 23, and CDR3 of SEQ ID NO: 24, and a light chain variable (VL) domain including: CDR1 of SEQ ID NO: 25, CDR2 of SEQ ID NO: 26, and CDR3 of SEQ ID NO: 27;
the antibody or antigen-binding fragment thereof that binds to the first WASp signature peptide includes: a heavy chain variable (VH) domain including CDR1 of SEQ ID NO: 28, CDR2 of SEQ ID NO: 29, and CDR3 of SEQ ID NO: 30, and a light chain variable (VL) domain including: CDR1 of SEQ ID NO: 31, CDR2 of SEQ ID NO: 32, and CDR3 of SEQ ID NO: 33; and/or
the antibody or antigen-binding fragment thereof that binds to the first BTK signature peptide includes: a heavy chain variable (VH) domain including CDR1 of SEQ ID NO: 34, CDR2 of SEQ ID NO: 35, and CDR3 of sequence GDI, and a light chain variable (VL) domain including: CDR1 of SEQ ID NO: 36, CDR2 of SEQ ID NO: 37, and CDR3 of SEQ ID NO: 38.
55. A method of any one of embodiments 42-44, wherein the endogenous ATP7B peptide is encoded by an amino acid sequence set forth in SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, or SEQ ID NO: 21.
56. A method of any one of embodiments 42-45, wherein the antibody or antigen-binding fragment thereof that binds to the endogenous ATP7B peptide includes: a heavy chain variable (VH) domain including CDR1 of SEQ ID NO: 57, CDR2 of SEQ ID NO: 58, and CDR3 of SEQ ID NO: 59, and a light chain variable (VL) domain including: CDR1 of SEQ ID NO: 60, CDR2 of SEQ ID NO: 61, and CDR3 of SEQ ID NO: 62.
57. A method of any one of embodiments 52-56, wherein each predetermined threshold ratio is calculated from the standard deviation of the mean ratio of each peptide concentration:endogenous ATP7B peptide concentration in a population of samples.
58. A method of any one of embodiments 52-57, wherein the method is performed as part of a newborn screening (NBS) that additionally screens the subject for one or more of phenylketonuria, primary congenital hypothyroidism, cystic fibrosis, and sickle cell disease.
59. A method of any one of embodiments 52-58, wherein the method is performed in the absence of clinical symptoms of SCID, WAS, and/or XLA in the subject.
60. A method of any one of embodiments 52-59, wherein the enzyme is trypsin.
61. A method of any one of embodiments 52-60, wherein the antibody or antigen-binding fragment thereof used for enrichment of the CD3ε signature peptide of SCID includes a VH domain of SEQ ID NO: 63.
62. A method of any one of embodiments 52-61, wherein the antibody or antigen-binding fragment thereof used for enrichment of the CD3ε signature peptide of SCID includes a VL domain of SEQ ID NO: 64.
63. A method of any one of embodiments 52-62, wherein the antibody or antigen-binding fragment thereof used for enrichment of the first WASp signature peptide of WAS includes a VH domain of SEQ ID NO: 65.
64. A method of any one of embodiments 52-63, wherein the antibody or antigen-binding fragment thereof used for enrichment of the first WASp signature peptide of WAS includes a VL domain of SEQ ID NO: 66.
65. A method of any one of embodiments 52-64, wherein the antibody or antigen-binding fragment thereof used for enrichment of the first WASp signature peptide of WAS includes a heavy chain of SEQ ID NO: 86.
66. A method of any one of embodiments 52-65, wherein the antibody or antigen-binding fragment thereof used for enrichment of the first WASp signature peptide of WAS includes a light chain of SEQ ID NO: 91.
67. A method of any one of embodiments 52-66, wherein the antibody or antigen-binding fragment thereof used for enrichment of the first BTK signature peptide of XLA includes a VH domain of SEQ ID NO: 67.
68. A method of any one of embodiments 52-67, wherein the antibody or antigen-binding fragment thereof used for enrichment of the first BTK signature peptide of XLA includes a VL domain of SEQ ID NO: 68.
69. A method of any one of embodiments 52-68, wherein the antibody or antigen-binding fragment thereof used for enrichment of the first BTK signature peptide of XLA includes a heavy chain of SEQ ID NO: 96.
70. The method of any one of embodiments 52-69, wherein the antibody or antigen-binding fragment thereof used for enrichment of the first BTK signature peptide of XLA includes a light chain of SEQ ID NO: 101.
71. A method of any one of embodiments 52-70, further including screening for cystinosis by enriching for:
a first CTNS signature peptide of cystinosis with an antibody or antigen-binding fragment thereof that binds to the first CTNS signature peptide;
a second CTNS signature peptide of cystinosis with an antibody or antigen-binding fragment thereof that binds to the second CTNS signature peptide;
a third CTNS signature peptide of cystinosis with an antibody or antigen-binding fragment thereof that binds to the third CTNS signature peptide;
a fourth CTNS signature peptide of cystinosis with an antibody or antigen-binding fragment thereof that binds to the fourth CTNS signature peptide;
a first SHPK signature peptide of cystinosis with an antibody or antigen-binding fragment thereof that binds to the first SHPK signature peptide;
a second SHPK signature peptide of cystinosis with an antibody or antigen-binding fragment thereof that binds to the second SHPK signature peptide; and/or
a third SHPK signature peptide of cystinosis with an antibody or antigen-binding fragment thereof that binds to the third SHPK signature peptide;
Calculating a ratio of signature peptide concentration:endogenous ATP7B peptide concentration for each signature peptide; and
Diagnosing the subject with cystinosis when the ratio of the first CTNS peptide concentration:endogenous ATP7B peptide concentration, the second CTNS peptide concentration:endogenous ATP7B peptide concentration, the third CTNS peptide concentration:endogenous ATP7B peptide concentration, the fourth CTNS peptide concentration:endogenous ATP7B peptide concentration, the first SHPK peptide concentration:endogenous ATP7B peptide concentration, the second SHPK peptide concentration:endogenous ATP7B peptide concentration, and/or the third SHPK signature peptide concentration:endogenous ATP7B peptide concentration is lower than a corresponding predetermined threshold ratio or when the first CTNS, the second CTNS, the third CTNS, the fourth CTNS, the first SHPK, the second SHPK, and/or the third SHPK signature peptides are absent.
72. A method of embodiment 71, wherein
the first CTNS signature peptide of cystinosis is encoded by an amino acid sequence set forth in SEQ ID NO: 6;
the second CTNS signature peptide of cystinosis is encoded by an amino acid sequence set forth in SEQ ID NO: 7 or 8;
the third CTNS signature peptide of cystinosis is encoded by an amino acid sequence set forth in SEQ ID NO: 12;
the fourth CTNS signature peptide of cystinosis is encoded by an amino acid sequence set forth in SEQ ID NO: 13;
the first SHPK signature peptide of cystinosis is encoded by an amino acid sequence set forth in SEQ ID NO: 9;
the second SHPK signature peptide of cystinosis is encoded by an amino acid sequence set forth in SEQ ID NO: 14; and/or
the third SHPK signature peptide of cystinosis is encoded by an amino acid sequence set forth in SEQ ID NO: 15.
73. A method of embodiment 71 or 72, wherein
the antibody or antigen-binding fragment thereof that binds to the first CTNS signature peptide of cystinosis includes: a heavy chain variable (VH) domain including CDR1 of SEQ ID NO: 39, CDR2 of SEQ ID NO: 40, and CDR3 of SEQ ID NO: 41, and a light chain variable (VL) domain including: CDR1 of SEQ ID NO: 42, CDR2 of SEQ ID NO: 43, and CDR3 of SEQ ID NO: 44;
the antibody or antigen-binding fragment thereof that binds to the second CTNS signature peptide includes: a heavy chain variable (VH) domain including CDR1 of SEQ ID NO: 45, CDR2 of SEQ ID NO: 46, and CDR3 of SEQ ID NO: 47, and a light chain variable (VL) domain including: CDR1 of SEQ ID NO: 48, CDR2 of SEQ ID NO: 49, and CDR3 of SEQ ID NO: 50; and/or
the antibody or antigen-binding fragment thereof that binds to the first SHPK signature peptide includes: a heavy chain variable (VH) domain including CDR1 of SEQ ID NO: 51, CDR2 of SEQ ID NO: 52, and CDR3 of SEQ ID NO: 53, and a light chain variable (VL) domain including: CDR1 of SEQ ID NO: 54, CDR2 of SEQ ID NO: 55, and CDR3 of SEQ ID NO: 56.
74. The method of any one of embodiments 71-73, wherein the antibody or antigen-binding fragment thereof used for enrichment of the first CTNS signature peptide of cystinosis of SEQ ID NO: 6 includes a VH domain of SEQ ID NO: 69.
75. A method of any one of embodiments 71-74, wherein the antibody or antigen-binding fragment thereof used for enrichment of the first CTNS signature peptide of cystinosis includes a VL domain of SEQ ID NO: 70.
76. A method of any one of embodiments 71-75, wherein the antibody or antigen-binding fragment thereof used for enrichment of the first CTNS signature peptide of cystinosis includes a heavy chain of SEQ ID NO: 106.
77. A method of any one of embodiments 71-76, wherein the antibody or antigen-binding fragment thereof used for enrichment of the first CTNS signature peptide of cystinosis includes a light chain of SEQ ID NO: 111.
78. A method of any one of embodiments 71-77, wherein the antibody or antigen-binding fragment thereof used for enrichment of the second CTNS signature peptide of cystinosis includes a VH domain of SEQ ID NO: 71.
79. A method of any one of embodiments 71-78, wherein the antibody or antigen-binding fragment thereof used for enrichment of the second CTNS signature peptide of cystinosis includes a VL domain of SEQ ID NO: 72.
80. A method of any one of embodiments 71-79, wherein the antibody or antigen-binding fragment thereof used for enrichment of the second CTNS signature peptide of cystinosis includes a heavy chain of SEQ ID NO: 116.
81. A method of any one of embodiments 71-80, wherein the antibody or antigen-binding fragment thereof used for enrichment of the second CTNS signature peptide of cystinosis includes a light chain of SEQ ID NO: 121.
82. A method of any one of embodiments 71-81, wherein the antibody or antigen-binding fragment thereof used for enrichment of the first SHPK signature peptide of cystinosis includes a VH domain of SEQ ID NO: 73.
83. A method of any one of embodiments 71-82, wherein the antibody or antigen-binding fragment thereof used for enrichment of the first SHPK signature peptide of cystinosis includes a VL domain of SEQ ID NO: 74.
84. A method of any one of embodiments 71-83, wherein the antibody or antigen-binding fragment thereof used for enrichment of the first SHPK signature peptide of cystinosis includes a heavy chain of SEQ ID NO: 126.
85. A method of any one of embodiments 71-84, wherein the antibody or antigen-binding fragment thereof used for enrichment of the first SHPK signature peptide of cystinosis includes a light chain of SEQ ID NO: 131.
86. A method of any one of embodiments 52-85, wherein the antibody or antigen-binding fragment thereof used for enrichment of the endogenous ATP7B peptide includes a VH domain of SEQ ID NO: 75.
87. A method of any one of embodiments 52-86, wherein the antibody or antigen-binding fragment thereof used for enrichment of the endogenous ATP7B signature peptide includes a VL domain of SEQ ID NO: 76.
88. A method of any one of embodiments 52-87, wherein the antibody or antigen-binding fragment thereof used for enrichment of the endogenous ATP7B signature peptide includes a heavy chain of SEQ ID NO: 136.
89. A method of any one of embodiments 52-88, wherein the antibody or antigen-binding fragment thereof used for enrichment of the endogenous ATP7B signature peptide includes a light chain of SEQ ID NO: 141.
90. A method of any one of embodiments 52-89, wherein the concentrations of the peptides are determined from corresponding known concentrations of reference signature peptides added prior to the LC-MRM-MS.
91. A method of detecting one or more signature peptides of severe combined immunodeficiency (SCID), Wiskott-Aldrich syndrome (WAS), and/or X-linked agammaglobulinemia (XLA) in one or more dried blood spot (DBS) samples, the method including:
Obtaining the one or more dried blood spot (DBS) samples;
Digesting proteins from blood of each DBS with an enzyme to yield one or more peptides;

Enriching for:

a CD3ε signature peptide of SCID with an antibody or antigen-binding fragment thereof that binds to the CD3ε signature peptide;
a first WASp signature peptide of WAS with an antibody or antigen-binding fragment thereof that binds to the first WASp signature peptide;
a second WASp signature peptide of WAS with an antibody or antigen-binding fragment thereof that binds to the second WASp signature peptide;
a first BTK signature peptide of XLA with an antibody or antigen-binding fragment thereof that binds to the first BTK signature peptide; and/or
a second BTK signature peptide with an antibody or antigen-binding fragment thereof that binds to the second BTK signature peptide;
Performing liquid chromatography-multiple reaction monitoring mass spectrometry (LC-MRM-MS) on the enriched peptides to determine concentrations of the peptides; and
Performing liquid chromatography-multiple reaction monitoring mass spectrometry (LC-MRM-MS) on the enriched peptides, thus detecting one or more signature peptides of SCID, WAS, and XLA in one or more DBS samples.
92. A method of embodiment 91, wherein
the CD3ε signature peptide of SCID is encoded by an amino acid sequence set forth in SEQ ID NO: 1;
the first WASp signature peptide of WAS is encoded by an amino acid sequence set forth in SEQ ID NO: 2;
the second WASp signature peptide of WAS is encoded by an amino acid sequence set forth in SEQ ID NO: 3;
the first BTK signature peptide of XLA is encoded by an amino acid sequence set forth in SEQ ID NO: 4; and/or
the second BTK signature peptide of XLA is encoded by an amino acid sequence set forth in SEQ ID NO: 5.
93. A method of embodiment 91 or 92, wherein
the antibody or antigen-binding fragment thereof that binds to the CD3ε signature peptide includes: a heavy chain variable (VH) domain including CDR1 of SEQ ID NO: 22, CDR2 of SEQ ID NO: 23, and CDR3 of SEQ ID NO: 24, and a light chain variable (VL) domain including: CDR1 of SEQ ID NO: 25, CDR2 of SEQ ID NO: 26, and CDR3 of SEQ ID NO: 27;
the antibody or antigen-binding fragment thereof that binds to the first WASp signature peptide includes: a heavy chain variable (VH) domain including CDR1 of SEQ ID NO: 28, CDR2 of SEQ ID NO: 29, and CDR3 of SEQ ID NO: 30, and a light chain variable (VL) domain including: CDR1 of SEQ ID NO: 31, CDR2 of SEQ ID NO: 32, and CDR3 of SEQ ID NO: 33; and/or
the antibody or antigen-binding fragment thereof that binds to the first BTK signature peptide includes: a heavy chain variable (VH) domain including CDR1 of SEQ ID NO: 34, CDR2 of SEQ ID NO: 35, and CDR3 of sequence GDI, and a light chain variable (VL) domain including: CDR1 of SEQ ID NO: 36, CDR2 of SEQ ID NO: 37, and CDR3 of SEQ ID NO: 38.
94. A method of any one of embodiments 91-93, wherein the enzyme is trypsin.
95. A method of any one of embodiments 91-94, wherein the antibody or antigen-binding fragment thereof used for enrichment of the CD3ε signature peptide of SCID includes a VH domain of SEQ ID NO: 63.
96. A method of any one of embodiments 91-95, wherein the antibody or antigen-binding fragment thereof used for enrichment of the CD3ε signature peptide of SCID includes a VL domain of SEQ ID NO: 64.
97. A method of any one of embodiments 91-96, wherein the antibody or antigen-binding fragment thereof used for enrichment of the first WASp signature peptide of WAS includes a VH domain of SEQ ID NO: 65.
98. A method of any one of embodiments 91-97, wherein the antibody or antigen-binding fragment thereof used for enrichment of the first WASp signature peptide of WAS includes a VL domain of SEQ ID NO: 66.
99. A method of any one of embodiments 91-98, wherein the antibody or antigen-binding fragment thereof used for enrichment of the first WASp signature peptide of WAS includes a heavy chain of SEQ ID NO: 86.
100. A method of any one of embodiments 91-99, wherein the antibody or antigen-binding fragment thereof used for enrichment of the first WASp signature peptide of WAS includes a light chain of SEQ ID NO: 91.
101. A method of any one of embodiments 91-100, wherein the antibody or antigen-binding fragment thereof used for enrichment of the first BTK signature peptide of XLA includes a VH domain of SEQ ID NO: 67.
102. A method of any one of embodiments 91-101, wherein the antibody or antigen-binding fragment thereof used for enrichment of the first BTK signature peptide of XLA includes a VL domain of SEQ ID NO: 68.
103. A method of any one of embodiments 91-102, wherein the antibody or antigen-binding fragment thereof used for enrichment of the first BTK signature peptide of XLA includes a heavy chain of SEQ ID NO: 96.
104. A method of any one of embodiments 91-103, wherein the antibody or antigen-binding fragment thereof used for enrichment of the first BTK signature peptide of XLA includes a light chain of SEQ ID NO: 101.
105. A method of any one of embodiments 91-104, further including detecting one or more signature peptides of cystinosis by enriching for:
a first CTNS signature peptide of cystinosis with an antibody or antigen-binding fragment thereof that binds to the first CTNS signature peptide;
a second CTNS signature peptide of cystinosis with an antibody or antigen-binding fragment thereof that binds to the second CTNS signature peptide;
a third CTNS signature peptide of cystinosis with an antibody or antigen-binding fragment thereof that binds to the third CTNS signature peptide;
a fourth CTNS signature peptide of cystinosis with an antibody or antigen-binding fragment thereof that binds to the fourth CTNS signature peptide;
a first SHPK signature peptide of cystinosis with an antibody or antigen-binding fragment thereof that binds to the first SHPK signature peptide;
a second SHPK signature peptide of cystinosis with an antibody or antigen-binding fragment thereof that binds to the second SHPK signature peptide; and/or
a third SHPK signature peptide of cystinosis with an antibody or antigen-binding fragment thereof that binds to the third SHPK signature peptide;
and
Performing liquid chromatography-multiple reaction monitoring mass spectrometry (LC-MRM-MS) on the enriched peptides, thus detecting one or more signature peptides of cystinosis in one or more DBS samples.
106. A method of embodiment 105, wherein
the first CTNS signature peptide of cystinosis is encoded by an amino acid sequence set forth in SEQ ID NO: 6;
the second CTNS signature peptide of cystinosis is encoded by an amino acid sequence set forth in SEQ ID NO: 7 or 8;
the third CTNS signature peptide of cystinosis is encoded by an amino acid sequence set forth in SEQ ID NO: 12;
the fourth CTNS signature peptide of cystinosis is encoded by an amino acid sequence set forth in SEQ ID NO: 13;
the first SHPK signature peptide of cystinosis is encoded by an amino acid sequence set forth in SEQ ID NO: 9;
the second SHPK signature peptide of cystinosis is encoded by an amino acid sequence set forth in SEQ ID NO: 14; and/or
the third SHPK signature peptide of cystinosis is encoded by an amino acid sequence set forth in SEQ ID NO: 15.
107. A method of embodiment 105 or 106, wherein
the antibody or antigen-binding fragment thereof that binds to the first CTNS signature peptide of cystinosis includes: a heavy chain variable (VH) domain including CDR1 of SEQ ID NO: 39, CDR2 of SEQ ID NO: 40, and CDR3 of SEQ ID NO: 41, and a light chain variable (VL) domain including: CDR1 of SEQ ID NO: 42, CDR2 of SEQ ID NO: 43, and CDR3 of SEQ ID NO: 44;
the antibody or antigen-binding fragment thereof that binds to the second CTNS signature peptide includes: a heavy chain variable (VH) domain including CDR1 of SEQ ID NO: 45, CDR2 of SEQ ID NO: 46, and CDR3 of SEQ ID NO: 47, and a light chain variable (VL) domain including: CDR1 of SEQ ID NO: 48, CDR2 of SEQ ID NO: 49, and CDR3 of SEQ ID NO: 50; and/or
the antibody or antigen-binding fragment thereof that binds to the first SHPK signature peptide includes: a heavy chain variable (VH) domain including CDR1 of SEQ ID NO: 51, CDR2 of SEQ ID NO: 52, and CDR3 of SEQ ID NO: 53, and a light chain variable (VL) domain including: CDR1 of SEQ ID NO: 54, CDR2 of SEQ ID NO: 55, and CDR3 of SEQ ID NO: 56.
108. A method of any one of embodiments 105-107, wherein the antibody or antigen-binding fragment thereof used for enrichment of the first CTNS signature peptide of cystinosis includes a VH domain of SEQ ID NO: 69.
109. A method of any one of embodiments 105-108, wherein the antibody or antigen-binding fragment thereof used for enrichment of the first CTNS signature peptide of cystinosis includes a VL domain of SEQ ID NO: 70.
110. A method of any one of embodiments 105-109, wherein the antibody or antigen-binding fragment thereof used for enrichment of the first CTNS signature peptide of cystinosis includes a heavy chain of SEQ ID NO: 106.
111. A method of any one of embodiments 105-110, wherein the antibody or antigen-binding fragment thereof used for enrichment of the first CTNS signature peptide of cystinosis includes a light chain of SEQ ID NO: 111.
112. A method of any one of embodiments 105-111, wherein the antibody or antigen-binding fragment thereof used for enrichment of the second CTNS signature peptide of cystinosis includes a VH domain of SEQ ID NO: 71.
113. A method of any one of embodiments 105-112, wherein the antibody or antigen-binding fragment thereof used for enrichment of the second CTNS signature peptide of cystinosis includes a VL domain of SEQ ID NO: 72.
114. A method of any one of embodiments 105-113, wherein the antibody or antigen-binding fragment thereof used for enrichment of the second CTNS signature peptide of cystinosis includes a heavy chain of SEQ ID NO: 116.
115. A method of any one of embodiments 105-114, wherein the antibody or antigen-binding fragment thereof used for enrichment of the second CTNS signature peptide of cystinosis includes a light chain of SEQ ID NO: 121.
116. A method of any one of embodiments 105-115, wherein the antibody or antigen-binding fragment thereof used for enrichment of the first SHPK signature peptide of cystinosis includes a VH domain of SEQ ID NO: 73.
117. A method of any one of embodiments 105-116, wherein the antibody or antigen-binding fragment thereof used for enrichment of the first SHPK signature peptide of cystinosis includes a VL domain of SEQ ID NO: 74.
118. A method of any one of embodiments 105-117, wherein the antibody or antigen-binding fragment thereof used for enrichment of the first SHPK signature peptide of cystinosis includes a heavy chain of SEQ ID NO: 126.
119. A method of any one of embodiments 105-118, wherein the antibody or antigen-binding fragment thereof used for enrichment of the first SHPK signature peptide of cystinosis includes a light chain of SEQ ID NO: 131.
120. A method of any one of embodiments 91-119, further including: Enriching for an endogenous ATP7B peptide with an antibody or antigen binding fragment thereof that binds to the endogenous ATP7B peptide; and Calculating a ratio of signature peptide concentration:endogenous ATP7B peptide concentration for each signature peptide.
121. A method of embodiment 120, wherein the endogenous ATP7B peptide is encoded by an amino acid sequence set forth in SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, or SEQ ID NO: 21.
122. A method of embodiment 120 or 121, wherein the antibody or antigen-binding fragment thereof that binds to the endogenous ATP7B peptide includes: a heavy chain variable (VH) domain including CDR1 of SEQ ID NO: 57, CDR2 of SEQ ID NO: 58, and CDR3 of SEQ ID NO: 59, and a light chain variable (VL) domain including: CDR1 of SEQ ID NO: 60, CDR2 of SEQ ID NO: 61, and CDR3 of SEQ ID NO: 62.
123. A method of any one of embodiments 91-122, further including detecting one or more signature peptides of Wilson Disease (WD) by enriching for:
a first ATP7B signature peptide with an antibody or antigen-binding fragment thereof that binds to the first ATP7B signature peptide;
a second ATP7B signature peptide with an antibody or antigen-binding fragment thereof that binds to the second ATP7B signature peptide;
a third ATP7B signature peptide with an antibody or antigen-binding fragment that binds to the third ATP7B signature peptide;
a fourth ATP7B signature peptide with an antibody or antigen-binding fragment thereof that binds to the fourth ATP7B signature peptide;
a fifth ATP7B signature peptide with an antibody or antigen-binding fragment thereof that binds to the fifth ATP7B signature peptide;
a sixth ATP7B signature peptide with an antibody or antigen-binding fragment thereof that binds to the sixth ATP7B signature peptide; and/or
a seventh ATP7B signature peptide with an antibody or antigen-binding fragment thereof;
Performing liquid chromatography-multiple reaction monitoring mass spectrometry (LC-MRM-MS) on the enriched peptides, thus detecting one or more signature peptides of WD in one or more DBS samples.
124. A method of embodiment 123, wherein
the first ATP7B signature peptide of WD is encoded by an amino acid sequence set forth in SEQ ID NO: 10;
the second ATP7B signature peptide of WD is encoded by an amino acid sequence set forth in SEQ ID NO: 11 or 21;
the third ATP7B signature peptide of WD is encoded by an amino acid sequence set forth in SEQ ID NO: 16;
the fourth ATP7B signature peptide of WD is encoded by an amino acid sequence set forth in SEQ ID NO: 17;
the fifth ATP7B signature peptide of WD is encoded by an amino acid sequence set forth in SEQ ID NO: 18;
the sixth ATP7B signature peptide of WD is encoded by an amino acid sequence set forth in SEQ ID NO: 19; and/or
the seventh ATP7B signature peptide of WD is encoded by an amino acid sequence set forth in SEQ ID NO: 20.
125. A method of embodiment 123 or 124, wherein the antibody or antigen-binding fragment thereof that binds to the second ATP7B signature peptide includes: a heavy chain variable (VH) domain including CDR1 of SEQ ID NO: 57, CDR2 of SEQ ID NO: 58, and CDR3 of SEQ ID NO: 59, and a light chain variable (VL) domain including: CDR1 of SEQ ID NO: 60, CDR2 of SEQ ID NO: 61, and CDR3 of SEQ ID NO: 62.
126. A method of any one of embodiments 123-125, wherein the antibody or antigen-binding fragment thereof used for enrichment of the second ATP7B signature peptide of WD includes a VH domain of SEQ ID NO: 75.
127. A method of any one of embodiments 123-126, wherein the antibody or antigen-binding fragment thereof used for enrichment of the second ATP7B signature peptide of WD includes a VL domain of SEQ ID NO: 76.
128. A method of any one of embodiments 123-127, wherein the antibody or antigen-binding fragment thereof used for enrichment of the second ATP7B signature peptide of WD includes a heavy chain of SEQ ID NO: 136.
129. A method of any one of embodiments 123-128, wherein the antibody or antigen-binding fragment thereof used for enrichment of the second ATP7B signature peptide of WD includes a light chain of SEQ ID NO: 141.
130. A method of any one of embodiments 91-129, further including comparing the concentration of each signature peptide to a corresponding predetermined threshold concentration.
131. A method of embodiment 130, wherein the corresponding predetermined threshold concentration is −1 standard deviation (SD), −1.1 SD, −1.2 SD, −1.3 SD, −1.4 SD, −1.5 SD, −1.6 SD, −1.7 SD, −1.8 SD, −1.9 SD, −2.0 SD, −2.1 SD, −2.2 SD, −2.3 SD, −2.4 SD, −2.5 SD, −2.6 SD, −2.7 SD, −2.8 SD, −2.9 SD, −3.0 SD, or more SD from the mean concentration of each signature peptide in a population of normal control subjects.
132. A method of embodiment 130 or 131, wherein the corresponding predetermined threshold concentration for each signature peptide is calculated from a standard deviation of the mean concentration of each signature peptide in a population of normal control subjects.
133. A method of embodiment 132, wherein the mean concentration of the CD3ε signature peptide in DBS from a population of normal control subjects includes a concentration in a range of 70 pmol/L to 400 pmol/L.
134. A method of embodiment 132 or 133, wherein the mean concentration of the first WASp signature peptide in DBS from a population of normal control subjects includes a concentration in a range of 600 pmol/L to 5000 pmol/L.
135. A method of any one of embodiments 132-134, wherein the mean concentration of the second WASp signature peptide in DBS from a population of normal control subjects includes a concentration in a range of 5500 pmol/L to 15000 pmol/L.
136. A method of any one of embodiments 132-135, wherein the mean concentration of the first BTK signature peptide in DBS from a population of normal control subjects includes a concentration in a range of 350 pmol/L to 2500 pmol/L.
137. A method of any one of embodiments 132-136, wherein the mean concentration of the second BTK signature peptide in DBS from a population of normal control subjects includes a concentration in a range of 550 pmol/L to 1600 pmol/L.
138. A method of any one of embodiments 132-137, wherein the mean concentration of the first CTNS signature peptide in DBS from a population of normal control subjects includes a concentration in a range of 40 pmol/L to 250 pmol/L.
139. A method of any one of embodiments 132-138, wherein the mean concentration of the first SHPK signature peptide in DBS from a population of normal control subjects includes a concentration in a range of 100 pmol/L to 8000 pmol/L.
140. A method of any one of embodiments 132-139, wherein the mean concentration of the second ATP7B signature peptide in DBS from a population of normal control subjects includes a concentration in a range of 90 pmol/L to 400 pmol/L.
141. A method of any one of embodiments 132-140, wherein the mean concentration of the third ATP7B signature peptide in DBS from a population of normal control subjects includes a concentration in a range of 30 pmol/L to 100 pmol/L.
142. A method of any one of embodiments 132-141, wherein the mean concentration of the seventh ATP7B signature peptide in DBS from a population of normal control subjects includes a concentration in a range of 200 pmol/L to 500 pmol/L.
143. A method of any one of embodiments 91-142, wherein the concentrations of the peptides are determined from corresponding known concentrations of reference signature peptides added prior to the LC-MRM-MS.
144. An assay for the screening of severe combined immunodeficiency (SCID), Wiskott-Aldrich syndrome (WAS), X-linked agammaglobulinemia (XLA), cystinosis, or Wilson Disease (WD) in a subject, the assay including:
(I) (i) an antibody or antigen-binding fragment thereof that binds to a CD3ε signature peptide of SCID;
(ii) an antibody or antigen-binding fragment thereof that binds to a first WASp signature peptide of WAS;
(iii) an antibody or antigen-binding fragment thereof that binds to a second WASp signature peptide of WAS;
(iv) an antibody or antigen-binding fragment thereof that binds to a first BTK signature peptide of XLA;
(v) an antibody or antigen-binding fragment thereof that binds to a second BTK signature peptide of XLA;
(vi) an antibody or antigen-binding fragment thereof that binds to a first CTNS signature peptide of cystinosis;
(vii) an antibody or antigen-binding fragment thereof that binds to a second CTNS signature peptide of cystinosis;
(viii) an antibody or antigen-binding fragment thereof that binds to a third CTNS signature peptide of cystinosis;
(ix) an antibody or antigen-binding fragment thereof that binds to a fourth CTNS signature peptide of cystinosis;
(x) an antibody or antigen-binding fragment thereof that binds to a first SHPK signature peptide of cystinosis;
(xi) an antibody or antigen-binding fragment thereof that binds to a second SHPK signature peptide of cystinosis;
(xii) an antibody or antigen-binding fragment thereof that binds to a third SHPK signature peptide of cystinosis;
(xiii) an antibody or antigen-binding fragment thereof that binds to a first ATP7B signature peptide of WD;
(xiv) an antibody or antigen-binding fragment thereof that binds to a second ATP7B signature peptide of WD;
(xv) an antibody or antigen-binding fragment thereof that binds to a third ATP7B signature peptide of WD;
(xvi) an antibody or antigen-binding fragment thereof that binds to a fourth ATP7B signature peptide of WD;
(xvii) an antibody or antigen-binding fragment thereof that binds to a fifth ATP7B signature peptide of WD;
(xviii) an antibody or antigen-binding fragment thereof that binds to a sixth ATP7B signature peptide of WD; and/or
(xix) an antibody or antigen-binding fragment thereof that binds to a seventh ATP7B signature peptide of WD;
and/or
(II) the corresponding reference signature peptides.
145. An assay of embodiment 144, wherein
(i) the CD3ε signature peptide and/or its reference signature peptide is encoded by an amino acid sequence set forth in SEQ ID NO: 1;
(ii) the first WASp signature peptide and/or its reference signature peptide is encoded by an amino acid sequence set forth in SEQ ID NO: 2;
(iii) the second WASp signature peptide and/or its reference signature peptide is encoded by an amino acid sequence set forth in SEQ ID NO: 3;
(iv) the first BTK signature peptide and/or its reference signature peptide is encoded by an amino acid sequence set forth in SEQ ID NO: 4; and/or
(v) the second BTK signature peptide and/or its reference signature peptide is encoded by an amino acid sequence set forth in SEQ ID NO: 5.
(vi) the first CTNS signature peptide and/or its reference signature peptide is encoded by an amino acid sequence set forth in SEQ ID NO: 6;
(vii) the second CTNS signature peptide and/or its reference signature peptide is encoded by an amino acid sequence set forth in SEQ ID NO: 7 or 8;
(viii) the third CTNS signature peptide and/or its reference signature peptide is encoded by an amino acid sequence set forth in SEQ ID NO: 12;
(ix) the fourth CTNS signature peptide and/or its reference signature peptide is encoded by an amino acid sequence set forth in SEQ ID NO: 13;
(x) the first SHPK signature peptide and/or its reference signature peptide is encoded by an amino acid sequence set forth in SEQ ID NO: 9;
(xi) the second SHPK signature peptide and/or its reference signature peptide is encoded by an amino acid sequence set forth in SEQ ID NO: 14;
(xii) the third SHPK signature peptide and/or its reference signature peptide is encoded by an amino acid sequence set forth in SEQ ID NO: 15;
(xiii) the first ATP7B signature peptide and/or its reference signature peptide is encoded by an amino acid sequence set forth in SEQ ID NO: 10;
(xiv) the second ATP7B signature peptide and/or its reference signature peptide is encoded by an amino acid sequence set forth in SEQ ID NO: 11 or 21;
(xv) the third ATP7B signature peptide and/or its reference signature peptide is encoded by an amino acid sequence set forth in SEQ ID NO: 16;
(xvi) the fourth ATP7B signature peptide and/or its reference signature peptide is encoded by an amino acid sequence set forth in SEQ ID NO: 17;
(xvii) the fifth ATP7B signature peptide and/or its reference signature peptide is encoded by an amino acid sequence set forth in SEQ ID NO: 18;
(xviii) the sixth ATP7B signature peptide and/or its reference signature peptide is encoded by an amino acid sequence set forth in SEQ ID NO: 19; and/or
(xix) the seventh ATP7B signature peptide and/or its reference signature peptide is encoded by an amino acid sequence set forth in SEQ ID NO: 20.
146. An assay of embodiment 144 or 145, wherein
the antibody or antigen-binding fragment thereof that binds to the CD3ε signature peptide includes: a heavy chain variable (VH) domain including CDR1 of SEQ ID NO: 22, CDR2 of SEQ ID NO: 23, and CDR3 of SEQ ID NO: 24, and a light chain variable (VL) domain including: CDR1 of SEQ ID NO: 25, CDR2 of SEQ ID NO: 26, and CDR3 of SEQ ID NO: 27;
the antibody or antigen-binding fragment thereof that binds to the first WASp signature peptide includes: a heavy chain variable (VH) domain including CDR1 of SEQ ID NO: 28, CDR2 of SEQ ID NO: 29, and CDR3 of SEQ ID NO: 30, and a light chain variable (VL) domain including: CDR1 of SEQ ID NO: 31, CDR2 of SEQ ID NO: 32, and CDR3 of SEQ ID NO: 33;
the antibody or antigen-binding fragment thereof that binds to the first BTK signature peptide includes: a heavy chain variable (VH) domain including CDR1 of SEQ ID NO: 34, CDR2 of SEQ ID NO: 35, and CDR3 of sequence GDI, and a light chain variable (VL) domain including: CDR1 of SEQ ID NO: 36, CDR2 of SEQ ID NO: 37, and CDR3 of SEQ ID NO: 38;
the antibody or antigen-binding fragment thereof that binds to the first CTNS signature peptide of cystinosis includes: a heavy chain variable (VH) domain including CDR1 of SEQ ID NO: 39, CDR2 of SEQ ID NO: 40, and CDR3 of SEQ ID NO: 41, and a light chain variable (VL) domain including: CDR1 of SEQ ID NO: 42, CDR2 of SEQ ID NO: 43, and CDR3 of SEQ ID NO: 44;
the antibody or antigen-binding fragment thereof that binds to the second CTNS signature peptide includes: a heavy chain variable (VH) domain including CDR1 of SEQ ID NO: 45, CDR2 of SEQ ID NO: 46, and CDR3 of SEQ ID NO: 47, and a light chain variable (VL) domain including: CDR1 of SEQ ID NO: 48, CDR2 of SEQ ID NO: 49, and CDR3 of SEQ ID NO: 50;
the antibody or antigen-binding fragment thereof that binds to the first SHPK signature peptide includes: a heavy chain variable (VH) domain including CDR1 of SEQ ID NO: 51, CDR2 of SEQ ID NO: 52, and CDR3 of SEQ ID NO: 53, and a light chain variable (VL) domain including: CDR1 of SEQ ID NO: 54, CDR2 of SEQ ID NO: 55, and CDR3 of SEQ ID NO: 56; and/or
the antibody or antigen-binding fragment thereof that binds to the second ATP7B signature peptide includes: a heavy chain variable (VH) domain including CDR1 of SEQ ID NO: 57, CDR2 of SEQ ID NO: 58, and CDR3 of SEQ ID NO: 59, and a light chain variable (VL) domain including: CDR1 of SEQ ID NO: 60, CDR2 of SEQ ID NO: 61, and CDR3 of SEQ ID NO: 62.
147. An assay of any one of embodiments 144-146, wherein the antibody or antigen-binding fragment thereof that binds to the CD3ε signature peptide of SCID includes a VH domain of SEQ ID NO: 63.
148. An assay of any one of embodiments 144-147, wherein the antibody or antigen-binding fragment thereof that binds to the CD3ε signature peptide of SCID includes a VL domain of SEQ ID NO: 64.
149. An assay of any one of embodiments 144-148, wherein the antibody or antigen-binding fragment thereof that binds to the first WASp signature peptide of WAS includes a VH domain of SEQ ID NO: 65.
150. An assay of any one of embodiments 144-149, wherein the antibody or antigen-binding fragment thereof that binds to the first WASp signature peptide of WAS includes a VL domain of SEQ ID NO: 66.
151. An assay of any one of embodiments 144-150, wherein the antibody or antigen-binding fragment thereof that binds to the first WASp signature peptide of WAS includes a heavy chain of SEQ ID NO: 86.
152. An assay of any one of embodiments 144-1551, wherein the antibody or antigen-binding fragment thereof that binds to the first WASp signature peptide of WAS includes a light chain of SEQ ID NO: 91.
153. An assay of any one of embodiments 144-152, wherein the antibody or antigen-binding fragment thereof that binds to the first BTK signature peptide of XLA includes a VH domain of SEQ ID NO: 67.
154. An assay of any one of embodiments 144-153, wherein the antibody or antigen-binding fragment thereof that binds to the first BTK signature peptide of XLA includes a VL domain of SEQ ID NO: 68.
155. An assay of any one of embodiments 144-154, wherein the antibody or antigen-binding fragment thereof that binds to the first BTK signature peptide of XLA includes a heavy chain of SEQ ID NO: 96.
156. An assay of any one of embodiments 144-155, wherein the antibody or antigen-binding fragment thereof that binds to the first BTK signature peptide of XLA includes a light chain of SEQ ID NO: 101.
157. An assay of any one of embodiments 144-156, wherein the antibody or antigen-binding fragment thereof that binds to the first CTNS signature peptide of cystinosis of SEQ ID NO: 6 includes a VH domain of SEQ ID NO: 69.
158. An assay of any one of embodiments 144-157, wherein the antibody or antigen-binding fragment thereof that binds to the first CTNS signature peptide of cystinosis of SEQ ID NO: 6 includes a VL domain of SEQ ID NO: 70.
159. An assay of any one of embodiments 144-158, wherein the antibody or antigen-binding fragment thereof that binds to the first CTNS signature peptide of cystinosis of SEQ ID NO: 6 includes a heavy chain of SEQ ID NO: 106.
160. An assay of any one of embodiments 144-159, wherein the antibody or antigen-binding fragment thereof that binds to the first CTNS signature peptide of cystinosis of SEQ ID NO: 6 includes a light chain of SEQ ID NO: 111.
161. An assay of any one of embodiments 144-160, wherein the antibody or antigen-binding fragment thereof that binds to the second CTNS signature peptide of cystinosis of SEQ ID NO: 7 or 8 includes a VH domain of SEQ ID NO: 71.
162. An assay of any one of embodiments 144-161, wherein the antibody or antigen-binding fragment thereof that binds to the second CTNS signature peptide of cystinosis of SEQ ID NO: 7 or 8 includes a VL domain of SEQ ID NO: 72.
163. An assay of any one of embodiments 144-162, wherein the antibody or antigen-binding fragment thereof that binds to the second CTNS signature peptide of cystinosis of SEQ ID NO: 7 or 8 includes a heavy chain of SEQ ID NO: 116.
164. An assay of any one of embodiments 144-163, wherein the antibody or antigen-binding fragment thereof that binds to the second CTNS signature peptide of cystinosis of SEQ ID NO: 7 or 8 includes a light chain of SEQ ID NO: 121.
165. An assay of any one of embodiments 144-164, wherein the antibody or antigen-binding fragment thereof that binds to the first SHPK signature peptide of cystinosis of SEQ ID NO: 9 includes a VH domain of SEQ ID NO: 73.
166. An assay of any one of embodiments 144-165, wherein the antibody or antigen-binding fragment thereof that binds to the first SHPK signature peptide of cystinosis of SEQ ID NO: 9 includes a VL domain of SEQ ID NO: 74.
167. An assay of any one of embodiments 144-166, wherein the antibody or antigen-binding fragment thereof that binds to the first SHPK signature peptide of cystinosis of SEQ ID NO: 9 includes a heavy chain of SEQ ID NO: 126.
168. An assay of any one of embodiments 144-167, wherein the antibody or antigen-binding fragment thereof that binds to the first SHPK signature peptide of cystinosis of SEQ ID NO: 9 includes a light chain of SEQ ID NO: 131.
169. An assay of any one of embodiments 144-168, wherein the antibody or antigen-binding fragment thereof that binds to the second ATP7B signature peptide of WD of SEQ ID NO: 11 or 21 includes a VH domain of SEQ ID NO: 75.
170. An assay of any one of embodiments 144-169, wherein the antibody or antigen-binding fragment thereof that binds to the second ATP7B signature peptide of WD of SEQ ID NO: 11 or 21 includes a VL domain of SEQ ID NO: 76.
171. An assay of any one of embodiments 144-170, wherein the antibody or antigen-binding fragment thereof that binds to the second ATP7B signature peptide of WD of SEQ ID NO: 11 or 21 includes a heavy chain of SEQ ID NO: 136.
172. An assay of any one of embodiments 144-171, wherein the antibody or antigen-binding fragment thereof that binds to the second ATP7B signature peptide of WD of SEQ ID NO: 11 or 21 includes a light chain of SEQ ID NO: 141.
173. An assay of any one of embodiments 144-172, wherein the reference signature peptides are isotopically labeled.
174. An assay of any one of embodiments 144-173, wherein the antibodies or antigen-binding fragments thereof are attached to magnetic beads.
175. An isolated antibody or antigen binding fragment thereof including: a heavy chain variable (VH) domain including CDR1 of SEQ ID NO: 22, CDR2 of SEQ ID NO: 23, and CDR3 of SEQ ID NO: 24, and a light chain variable (VL) domain including: CDR1 of SEQ ID NO: 25, CDR2 of SEQ ID NO: 26, and CDR3 of SEQ ID NO: 27.
176. The isolated antibody or antigen binding fragment thereof of embodiment 175, wherein the VH domain includes SEQ ID NO: 63.
177. The isolated antibody or antigen binding fragment thereof of embodiment 175 or 176, wherein the VL domain includes SEQ ID NO: 64.
178. An isolated antibody or antigen binding fragment thereof including: a heavy chain variable (VH) domain including CDR1 of SEQ ID NO: 28, CDR2 of SEQ ID NO: 29, and CDR3 of SEQ ID NO: 30, and a light chain variable (VL) domain including: CDR1 of SEQ ID NO: 31, CDR2 of SEQ ID NO: 32, and CDR3 of SEQ ID NO: 33.
179. The isolated antibody or antigen binding fragment thereof of embodiment 178, wherein the VH domain includes SEQ ID NO: 65.
180. The isolated antibody or antigen binding fragment thereof of embodiment 178 or 179, wherein the VL domain includes SEQ ID NO: 66.
181. The isolated antibody or antigen binding fragment thereof of any one of embodiments 178-180, wherein the heavy chain includes SEQ ID NO: 86.
182. The isolated antibody or antigen binding fragment thereof of any one of embodiments 178-181, wherein the light chain includes SEQ ID NO: 91.
183. An isolated antibody or antigen binding fragment thereof including: a heavy chain variable (VH) domain including CDR1 of SEQ ID NO: 34, CDR2 of SEQ ID NO: 35, and CDR3 of sequence GDI, and a light chain variable (VL) domain including: CDR1 of SEQ ID NO: 36, CDR2 of SEQ ID NO: 37, and CDR3 of SEQ ID NO: 38.
184. The isolated antibody or antigen binding fragment thereof of embodiment 183, wherein the VH domain includes SEQ ID NO: 67.
185. The isolated antibody or antigen binding fragment thereof of embodiment 183 or 184, wherein the VL domain includes SEQ ID NO: 68.
186. The isolated antibody or antigen binding fragment thereof of any one of embodiments 183-185, wherein the heavy chain includes SEQ ID NO: 96.
187. The isolated antibody or antigen binding fragment thereof of any one of embodiments 183-186, wherein the light chain includes SEQ ID NO: 101.
188. An isolated antibody or antigen binding fragment thereof including: a heavy chain variable (VH) domain including CDR1 of SEQ ID NO: 39, CDR2 of SEQ ID NO: 40, and CDR3 of SEQ ID NO: 41, and a light chain variable (VL) domain including: CDR1 of SEQ ID NO: 42, CDR2 of SEQ ID NO: 43, and CDR3 of SEQ ID NO: 44.
189. The isolated antibody or antigen binding fragment thereof of embodiment 188, wherein the VH domain includes SEQ ID NO: 69.
190. The isolated antibody or antigen binding fragment thereof of embodiment 188 or 189, wherein the VL domain includes SEQ ID NO: 70.
191. The isolated antibody or antigen binding fragment thereof of any one of embodiments 188-190, wherein the heavy chain includes SEQ ID NO: 106.
192. The isolated antibody or antigen binding fragment thereof of any one of embodiments 188-191, wherein the light chain includes SEQ ID NO: 111.
193. An isolated antibody or antigen binding fragment thereof including: a heavy chain variable (VH) domain including CDR1 of SEQ ID NO: 45, CDR2 of SEQ ID NO: 46, and CDR3 of SEQ ID NO: 47, and a light chain variable (VL) domain including: CDR1 of SEQ ID NO: 48, CDR2 of SEQ ID NO: 49, and CDR3 of SEQ ID NO: 50.
194. The isolated antibody or antigen binding fragment thereof of embodiment 193, wherein the VH domain includes SEQ ID NO: 71.
195. The isolated antibody or antigen binding fragment thereof of embodiment 193 or 194, wherein the VL domain includes SEQ ID NO: 72.
196. The isolated antibody or antigen binding fragment thereof of any one of embodiments 193-195, wherein the heavy chain includes SEQ ID NO: 116.
197. The isolated antibody or antigen binding fragment thereof of any one of embodiments 193-196, wherein the light chain includes SEQ ID NO: 121.
198. An isolated antibody or antigen binding fragment thereof including: a heavy chain variable (VH) domain including CDR1 of SEQ ID NO: 51, CDR2 of SEQ ID NO: 52, and CDR3 of SEQ ID NO: 53, and a light chain variable (VL) domain including: CDR1 of SEQ ID NO: 54, CDR2 of SEQ ID NO: 55, and CDR3 of SEQ ID NO: 56.
199. The isolated antibody or antigen binding fragment thereof of embodiment 198, wherein the VH domain includes SEQ ID NO: 73.
200. The isolated antibody or antigen binding fragment thereof of embodiment 198 or 199, wherein the VL domain includes SEQ ID NO: 74.
201. The isolated antibody or antigen binding fragment thereof of any one of embodiments 198-200, wherein the heavy chain includes SEQ ID NO: 126.
202. The isolated antibody or antigen binding fragment thereof of any one of embodiments 198-201, wherein the light chain includes SEQ ID NO: 131.
203. An isolated antibody or antigen binding fragment thereof including: a heavy chain variable (VH) domain including CDR1 of SEQ ID NO: 57, CDR2 of SEQ ID NO: 58, and CDR3 of SEQ ID NO: 59, and a light chain variable (VL) domain including: CDR1 of SEQ ID NO: 60, CDR2 of SEQ ID NO: 61, and CDR3 of SEQ ID NO: 62.
204. The isolated antibody or antigen binding fragment thereof of embodiment 203, wherein the VH domain includes SEQ ID NO: 75.
205. The isolated antibody or antigen binding fragment thereof of embodiment 203 or 204, wherein the VL domain includes SEQ ID NO: 76.
206. The isolated antibody or antigen binding fragment thereof of any one of embodiments 203-205, wherein the heavy chain includes SEQ ID NO: 136.
207. The isolated antibody or antigen binding fragment thereof of any one of embodiments 203-206, wherein the light chain includes SEQ ID NO: 141.
208. A kit including:
(I) (i) an antibody or antigen-binding fragment thereof that binds to a CD3ε signature peptide of SCID;
(ii) an antibody or antigen-binding fragment thereof that binds to a first WASp signature peptide of WAS;
(iii) an antibody or antigen-binding fragment thereof that binds to a second WASp signature peptide of WAS;
(iv) an antibody or antigen-binding fragment thereof that binds to a first BTK signature peptide of XLA;
(v) an antibody or antigen-binding fragment thereof that binds to a second BTK signature peptide of XLA;
(vi) an antibody or antigen-binding fragment thereof that binds to a first CTNS signature peptide of cystinosis;
(vii) an antibody or antigen-binding fragment thereof that binds to a second CTNS signature peptide of cystinosis;
(viii) an antibody or antigen-binding fragment thereof that binds to a third CTNS signature peptide of cystinosis;
(ix) an antibody or antigen-binding fragment thereof that binds to a fourth CTNS signature peptide of cystinosis;
(x) an antibody or antigen-binding fragment thereof that binds to a first SHPK signature peptide of cystinosis;
(xi) an antibody or antigen-binding fragment thereof that binds to a second SHPK signature peptide of cystinosis;
(xii) an antibody or antigen-binding fragment thereof that binds to a third SHPK signature peptide of cystinosis;
(xiii) an antibody or antigen-binding fragment thereof that binds to a first ATP7B signature peptide of WD;
(xiv) an antibody or antigen-binding fragment thereof that binds to a second ATP7B signature peptide of WD;
(xv) an antibody or antigen-binding fragment thereof that binds to a third ATP7B signature peptide of WD;
(xvi) an antibody or antigen-binding fragment thereof that binds to a fourth ATP7B signature peptide of WD;
(xvii) an antibody or antigen-binding fragment thereof that binds to a fifth ATP7B signature peptide of WD;
(xviii) an antibody or antigen-binding fragment thereof that binds to a sixth ATP7B signature peptide of WD; and/or
(xix) an antibody or antigen-binding fragment thereof that binds to a seventh ATP7B signature peptide of WD;
and/or
(II) the corresponding reference signature peptides; and
(III) use instructions on use of contents in the kit.
209. A kit of embodiment 208, wherein
(i) the CD3ε signature peptide and/or its reference signature peptide is encoded by an amino acid sequence set forth in SEQ ID NO: 1;
(ii) the first WASp signature peptide and/or its reference signature peptide is encoded by an amino acid sequence set forth in SEQ ID NO: 2;
(iii) the second WASp signature peptide and/or its reference signature peptide is encoded by an amino acid sequence set forth in SEQ ID NO: 3;
(iv) the first BTK signature peptide and/or its reference signature peptide is encoded by an amino acid sequence set forth in SEQ ID NO: 4; and/or
(v) the second BTK signature peptide and/or its reference signature peptide is encoded by an amino acid sequence set forth in SEQ ID NO: 5.
(vi) the first CTNS signature peptide and/or its reference signature peptide is encoded by an amino acid sequence set forth in SEQ ID NO: 6;
(vii) the second CTNS signature peptide and/or its reference signature peptide is encoded by an amino acid sequence set forth in SEQ ID NO: 7 or 8;
(viii) the third CTNS signature peptide and/or its reference signature peptide is encoded by an amino acid sequence set forth in SEQ ID NO: 12;
(ix) the fourth CTNS signature peptide and/or its reference signature peptide is encoded by an amino acid sequence set forth in SEQ ID NO: 13;
(x) the first SHPK signature peptide and/or its reference signature peptide is encoded by an amino acid sequence set forth in SEQ ID NO: 9;
(xi) the second SHPK signature peptide and/or its reference signature peptide is encoded by an amino acid sequence set forth in SEQ ID NO: 14;
(xii) the third SHPK signature peptide and/or its reference signature peptide is encoded by an amino acid sequence set forth in SEQ ID NO: 15;
(xiii) the first ATP7B signature peptide and/or its reference signature peptide is encoded by an amino acid sequence set forth in SEQ ID NO: 10;
(xiv) the second ATP7B signature peptide and/or its reference signature peptide is encoded by an amino acid sequence set forth in SEQ ID NO: 11 or 21;
(xv) the third ATP7B signature peptide and/or its reference signature peptide is encoded by an amino acid sequence set forth in SEQ ID NO: 16;
(xvi) the fourth ATP7B signature peptide and/or its reference signature peptide is encoded by an amino acid sequence set forth in SEQ ID NO: 17;
(xvii) the fifth ATP7B signature peptide and/or its reference signature peptide is encoded by an amino acid sequence set forth in SEQ ID NO: 18;
(xviii) the sixth ATP7B signature peptide and/or its reference signature peptide is encoded by an amino acid sequence set forth in SEQ ID NO: 19; and/or
(xix) the seventh ATP7B signature peptide and/or its reference signature peptide is encoded by an amino acid sequence set forth in SEQ ID NO: 20.
210. A kit of embodiment 208 or 209, wherein
the antibody or antigen-binding fragment thereof that binds to the CD3ε signature peptide includes: a heavy chain variable (VH) domain including CDR1 of SEQ ID NO: 22, CDR2 of SEQ ID NO: 23, and CDR3 of SEQ ID NO: 24, and a light chain variable (VL) domain including: CDR1 of SEQ ID NO: 25, CDR2 of SEQ ID NO: 26, and CDR3 of SEQ ID NO: 27;
the antibody or antigen-binding fragment thereof that binds to the first WASp signature peptide includes: a heavy chain variable (VH) domain including CDR1 of SEQ ID NO: 28, CDR2 of SEQ ID NO: 29, and CDR3 of SEQ ID NO: 30, and a light chain variable (VL) domain including: CDR1 of SEQ ID NO: 31, CDR2 of SEQ ID NO: 32, and CDR3 of SEQ ID NO: 33;
the antibody or antigen-binding fragment thereof that binds to the first BTK signature peptide includes: a heavy chain variable (VH) domain including CDR1 of SEQ ID NO: 34, CDR2 of SEQ ID NO: 35, and CDR3 of sequence GDI, and a light chain variable (VL) domain including: CDR1 of SEQ ID NO: 36, CDR2 of SEQ ID NO: 37, and CDR3 of SEQ ID NO: 38;
the antibody or antigen-binding fragment thereof that binds to the first CTNS signature peptide of cystinosis includes: a heavy chain variable (VH) domain including CDR1 of SEQ ID NO: 39, CDR2 of SEQ ID NO: 40, and CDR3 of SEQ ID NO: 41, and a light chain variable (VL) domain including: CDR1 of SEQ ID NO: 42, CDR2 of SEQ ID NO: 43, and CDR3 of SEQ ID NO: 44;
the antibody or antigen-binding fragment thereof that binds to the second CTNS signature peptide includes: a heavy chain variable (VH) domain including CDR1 of SEQ ID NO: 45, CDR2 of SEQ ID NO: 46, and CDR3 of SEQ ID NO: 47, and a light chain variable (VL) domain including: CDR1 of SEQ ID NO: 48, CDR2 of SEQ ID NO: 49, and CDR3 of SEQ ID NO: 50;
the antibody or antigen-binding fragment thereof that binds to the first SHPK signature peptide includes: a heavy chain variable (VH) domain including CDR1 of SEQ ID NO: 51, CDR2 of SEQ ID NO: 52, and CDR3 of SEQ ID NO: 53, and a light chain variable (VL) domain including: CDR1 of SEQ ID NO: 54, CDR2 of SEQ ID NO: 55, and CDR3 of SEQ ID NO: 56; and/or
the antibody or antigen-binding fragment thereof that binds to the second ATP7B signature peptide includes: a heavy chain variable (VH) domain including CDR1 of SEQ ID NO: 57, CDR2 of SEQ ID NO: 58, and CDR3 of SEQ ID NO: 59, and a light chain variable (VL) domain including: CDR1 of SEQ ID NO: 60, CDR2 of SEQ ID NO: 61, and CDR3 of SEQ ID NO: 62.
211. A kit of any one of embodiments 208-210, wherein the antibody or antigen-binding fragment thereof that binds to the CD3ε signature peptide of SCID includes a VH domain of SEQ ID NO: 63.
212. A kit of any one of embodiments 208-211, wherein the antibody or antigen-binding fragment thereof that binds to the CD3ε signature peptide of SCID includes a VL domain of SEQ ID NO: 64.
213. A kit of any one of embodiments 208-212, wherein the antibody or antigen-binding fragment thereof that binds to the first WASp signature peptide of WAS includes a VH domain of SEQ ID NO: 65.
214. A kit of any one of embodiments 208-213, wherein the antibody or antigen-binding fragment thereof that binds to the first WASp signature peptide of WAS includes a VL domain of SEQ ID NO: 66.
215. A kit of any one of embodiments 208-214, wherein the antibody or antigen-binding fragment thereof that binds to the first WASp signature peptide of WAS includes a heavy chain of SEQ ID NO: 86.
216. A kit of any one of embodiments 208-215, wherein the antibody or antigen-binding fragment thereof that binds to the first WASp signature peptide of WAS includes a light chain of SEQ ID NO: 91.
217. A kit of any one of embodiments 208-216, wherein the antibody or antigen-binding fragment thereof that binds to the first BTK signature peptide of XLA includes a VH domain of SEQ ID NO: 67.
218. A kit of any one of embodiments 208-217, wherein the antibody or antigen-binding fragment thereof that binds to the first BTK signature peptide of XLA includes a VL domain of SEQ ID NO: 68.
219. A kit of any one of embodiments 208-218, wherein the antibody or antigen-binding fragment thereof that binds to the first BTK signature peptide of XLA includes a heavy chain of SEQ ID NO: 96.
220. A kit of any one of embodiments 208-219, wherein the antibody or antigen-binding fragment thereof that binds to the first BTK signature peptide of XLA includes a light chain of SEQ ID NO: 101.
221. A kit of any one of embodiments 208-220, wherein the antibody or antigen-binding fragment thereof that binds to the first CTNS signature peptide of cystinosis of SEQ ID NO: 6 includes a VH domain of SEQ ID NO: 69.
222. A kit of any one of embodiments 208-221, wherein the antibody or antigen-binding fragment thereof that binds to the first CTNS signature peptide of cystinosis of SEQ ID NO: 6 includes a VL domain of SEQ ID NO: 70.
223. A kit of any one of embodiments 208-222, wherein the antibody or antigen-binding fragment thereof that binds to the first CTNS signature peptide of cystinosis of SEQ ID NO: 6 includes a heavy chain of SEQ ID NO: 106.
224. A kit of any one of embodiments 208-223, wherein the antibody or antigen-binding fragment thereof that binds to the first CTNS signature peptide of cystinosis of SEQ ID NO: 6 includes a light chain of SEQ ID NO: 111.
225. A kit of any one of embodiments 208-224, wherein the antibody or antigen-binding fragment thereof that binds to the second CTNS signature peptide of cystinosis of SEQ ID NO: 7 or 8 includes a VH domain of SEQ ID NO: 71.
226. A kit of any one of embodiments 208-225, wherein the antibody or antigen-binding fragment thereof that binds to the second CTNS signature peptide of cystinosis of SEQ ID NO: 7 or 8 includes a VL domain of SEQ ID NO: 72.
227. A kit of any one of embodiments 208-226, wherein the antibody or antigen-binding fragment thereof that binds to the second CTNS signature peptide of cystinosis of SEQ ID NO: 7 or 8 includes a heavy chain of SEQ ID NO: 116.
228. A kit of any one of embodiments 208-227, wherein the antibody or antigen-binding fragment thereof that binds to the second CTNS signature peptide of cystinosis of SEQ ID NO: 7 or 8 includes a light chain of SEQ ID NO: 121.
229. A kit of any one of embodiments 208-228, wherein the antibody or antigen-binding fragment thereof that binds to the first SHPK signature peptide of cystinosis of SEQ ID NO: 9 includes a VH domain of SEQ ID NO: 73.
230. A kit of any one of embodiments 208-229, wherein the antibody or antigen-binding fragment thereof that binds to the first SHPK signature peptide of cystinosis of SEQ ID NO: 9 includes a VL domain of SEQ ID NO: 74.
231. A kit of any one of embodiments 208-230, wherein the antibody or antigen-binding fragment thereof that binds to the first SHPK signature peptide of cystinosis of SEQ ID NO: 9 includes a heavy chain of SEQ ID NO: 126.
232. A kit of any one of embodiments 208-231, wherein the antibody or antigen-binding fragment thereof that binds to the first SHPK signature peptide of cystinosis of SEQ ID NO: 9 includes a light chain of SEQ ID NO: 131.
233. A kit of any one of embodiments 208-232, wherein the antibody or antigen-binding fragment thereof that binds to the second ATP7B signature peptide of WD of SEQ ID NO: 11 or 21 includes a VH domain of SEQ ID NO: 75.
234. A kit of any one of embodiments 208-233, wherein the antibody or antigen-binding fragment thereof that binds to the second ATP7B signature peptide of WD of SEQ ID NO: 11 or 21 includes a VL domain of SEQ ID NO: 76.
235. A kit of any one of embodiments 208-234, wherein the antibody or antigen-binding fragment thereof that binds to the second ATP7B signature peptide of WD of SEQ ID NO: 11 or 21 includes a heavy chain of SEQ ID NO: 136.
236. A kit of any one of embodiments 208-235, wherein the antibody or antigen-binding fragment thereof that binds to the second ATP7B signature peptide of WD of SEQ ID NO: 11 or 21 includes a light chain of SEQ ID NO: 141.
237. A kit of any one of embodiments 208-236, further including one or more of filter paper card, punch tool, digestion enzymes, digestion buffers, solid support for the antibodies or antigen-binding fragments thereof; and elution buffers.
238. A kit of any one of embodiments 208-237, wherein the reference signature peptides are isotopically labeled.
239. A kit of any one of embodiments 208-238, wherein the antibodies or antigen-binding fragments thereof are attached to magnetic beads.

(XI) EXPERIMENTAL EXAMPLES

Example 1. Summary. A study was undertaken to evaluate whether a multiplex assay based on peptide immunoaffinity enrichment coupled with selected reaction monitoring mass spectrometry (immuno-SRM) can reliably and precisely distinguish affected patients with CD3ε-associated severe combined immunodeficiency (SCID), Wiskott-Aldrich Syndrome (WAS), and X-linked agammaglobulinemia (XLA) from one another and from unaffected normal control dried blood spot (DBS) samples. A blinded, multiplexed analysis of proteolytically-generated peptides from CD3ε, WASp, and BTK (for SCID, WAS, and XLA, respectively) in DBS samples from 42 primary immunodeficiency disorders (PIDD) patients, 40 normal adult controls, and 62 normal newborns was performed. The immuno-SRM assays reliably quantified the target peptides in DBS, including intra- and inter-assay precision (11-22% and 11-43%), linearity (1.39-2000 fmol peptide), and stability (≤0.09% difference in 72 h). Analysis of signature peptides found a statistically significant reduction (or absence) of peptide levels in affected patients compared to control groups (SCID: p=0.05, WAS and BTK: p=0.0001). Immuno-SRM-based quantification of proteotypic peptides from CD3ε, WASp, and BTK in DBS distinguishes relevant PIDD cases from controls. The approach can be employed to conduct large-scale multiplexed newborn screening of selective PIDDs.
Materials and Methods. Patient Samples. PIDD and normal control blood samples were obtained from Seattle Children's Immunology Diagnostic Laboratory. Newborn DBS were retrieved from the Washington State Newborn Screening Laboratory (Shoreline, Wash.) after Institutional Review Board approval. XLA DBS were collected from 20 suspected Vietnamese patients and shipped per regular mail to Seattle Children's Hospital. Genotypes of these patients by Sanger sequencing was previously reported in Segundo et al. Front Immunol. Frontiers; 2018; 9: 289. In total, DBS samples from 42 PIDD patients and 40 normal controls were obtained. Normal control and PIDD patient DBS were prepared by pipetting 70 μL of blood/12 mm spot onto filter paper cards (Protein Saver 903 Card, Whatman, Piscataway, N.J.), allowed to dry at room temperature overnight, and stored in sealed plastic bags at −80° C. until use. Affected patient samples were shipped from collection locations and stored at −80° C. until use.
Selection of Surrogate Peptides and Antibody Production. Surrogate peptides for CD3ε, WASp, and BTK were selected by in silico trypsin digestion and NCBI BLAST tools. Final peptide selections were made according to accepted major criteria for immuno-SRM development including peptide length, lack of post-transcriptional modifications, and uniqueness in the human genome by BLAST searching as previously described. Kerfoot et al. Proteomics Clin Appl. 2012; 6: 394-402; Abbatiello et al. Mol. Cell Proteomics. American Society for Biochemistry and Molecular Biology; 2015; 14: 2357-2374; Hoofnagle et al. Clin. Chem. 2016; 62: 48-69. Peptide selection and monoclonal antibody production for ATP7B signature peptides have been previously reported. Jung et al. 2017, supra. Crude peptides were then screened empirically to determine suitability for detection and quantification by liquid chromatography-tandem mass spectrometry (LC-MS/MS).
Affinity-purified rabbit polyclonal antibodies (pAb), or monoclonal antibodies (mAb), were successfully generated against five peptides by Pacific Immunology (Ramona, Calif.) or Fred Hutchinson Cancer Research Center Immunology lab (Seattle, Wash.). Briefly, signature peptides were synthesized with a C-terminal extension (GSGC, SEQ ID NO: 77) or an N-terminal cysteine extension and conjugated to keyhole limpet hemocyanin (KHL) for immunization. Two New Zealand white rabbits or five mice were injected per peptide. pAbs or mAbs for all selected peptides successfully underwent affinity-purification from 25 mL of antiserum.
Immuno-SRM Assay Reagents. ProteaseMAX™ Surfactant (no. V2072) and proteomics grade trypsin (no. V5113) were purchased from Promega (Madison, Wis.). Bovine serum albumin standard (200 mg/m L), and (3-[3-cholamidopropyl)=dimethylammonio]-1-propanesulfonate) (Pierce™ CHAPS, no. P128300) detergent were obtained from Thermo Fisher Scientific (Waltham, Mass.). Ammonium bicarbonate (40867-50G-F) was purchased from Fluka Analytical (Munich, Germany). Acetonitrile (no. A955), water (no. W6, LCMS optima grade), formic acid (no. P128905), and phosphate-buffered saline (PBS, no. 10010-023) were obtained from Thermo Fisher Scientific (Waltham, Mass.).
Heavy stable isotope-labeled peptides were obtained from Anaspec (Fremont, Calif.). The stable isotope-labeled peptides were purified >95% by HPLC and the C-terminal arginine or lysine was labeled with ¹³C and ¹⁵N atoms, resulting in a mass shift of +8 or +10 Da, respectively. Aliquots were stored in 5% acetonitrile/0.1% formic acid at −20° C. until use.
Antibodies were immobilized on 2.8 μm Dynabeads Protein G magnetic beads (no. 10004D, Invitrogen, Carlsbad, Calif.) in a 1 μg antibody-to-2.5 μL of beads ratio. In brief, 250 μL of the beads were added to 1.5 mL Eppendorf tubes (022363204 Eppendorf) and washed twice with 250 μL of 1×PBS, followed by the addition of 100 μg of antibody and 1×PBS+0.03% CHAPS (no. 28300, Thermo Scientific, Waltham, Mass.) to yield a total 250 μL of volume. The antibodies were allowed to couple to the beads overnight with tumbling at 4° C. The next day, the antibodies were immobilized onto the beads with chemical cross-linking. Briefly, antibody beads were collected using magnetic pulldown, excess PBS was discarded, and 300 μL of freshly prepared 20 mM DMP (dimethyl pimelimidate dihydrochloride, no. D8388, Sigma Aldrich, St. Louis, Mo.) in 200 mM triethanolamine, pH 8.5 (no. T58300, Sigma Aldrich, St. Louis, Mo.) was added. The samples were tumbled for 30 min at room temperature, and the DMP in triethanolamine was discarded. 250 μL of 150 mM monoethanolamine (no. 411000, Sigma Aldrich, St. Louis, Mo.) was added and the beads were tumbled at room temperature for 30 min. The antibody beads were washed twice using 250 μL of 5% acetic acid+0.03% CHAPS (5 min of tumbling at room temperate each time), and washed once more using 250 μL of 1×PBS+0.03% CHAPS. The CD3ε, WASp, and BTK antibody-linked beads were then washed and incubated in 5% acetic acid +3% acetonitrile (ACN), washed with 250 μL of 1×PBS+0.03% CHAPS, and the latter two steps were repeated once. All antibody-linked beads were washed with 250 μL of 1×PBS+0.03% CHAPS until neutral pH (7.0) was achieved. The washed antibody-linked beads were then resuspended in 250 μL of 1×PBS+0.03% CHAPS and 2.5 μL of NaN₃(52002-5G Sigma Aldrich) for anti-fungal properties and stored at 4° C. until use.
DBS Protein Extraction and Trypsin Digestion. For each sample (blinded normal controls or patients), one entire DBS spot (13 mm) containing 70 μL blood was perforated into 17 punches at 3-mm diameter with a standard leather punch tool. Final sample representation was SCID: n=3, WAS: n=11, XLA: n=26, and normal controls (n=40). The punches were placed in a 1.5 mL eppendorf tube, and 490 μL of 0.1% ProteaseMax™ in 50 mM ammonium biocarbonate (pH 8) was added into each tube. The tubes were vortexed for 1 h on the Eppendorf MixMate (Eppendorf, Hamburg, Germany), after which 10 μL of each sample were aliquoted and diluted 200-fold for Bradford assay to determine protein concentration. Disulfide bond reduction was performed with 2 M DTT at 5 mM, and an additional 490 μL of 0.1% ProteaseMax™ in 50 mM ammonium biocarbonate (pH 8) was added into each tube before incubation in 37° C. water bath for 30 minutes. Trypsin was then added at a 1:50 enzyme to protein ratio (w/w), and acetonitrile was added to a final concentration of 15%. The mixture was incubated in a 37° C. water bath overnight for digestion before centrifugation for 10 minutes at 13,000 RPM before each supernatant was transferred to a new tube and dried in the Savant™ SpeedVac™ High Capacity Concentrator (Thermo Fisher Scientific, Waltham, Mass.). All trypsinized DBS digests were stored at −80° C. until use.
For samples analyzed from the Washington State NBS laboratory, 5 or 6 3-mm punches were used for protein extraction and digestion (n=62). Procedures were identical to those for previous samples except that volumes were reduced as follows: 150 μL of 0.1% ProteaseMax™ and 0.78 μL DTT for each addition.
Peptide Immunoaffinity Enrichment. DBS digests were resuspended in 1×PBS+0.03% CHAPS to yield a 1 μg/μL nominal protein digest concentration. Cross-linked, antibody-coated beads were added to a total mass of 2 μg pAb for each target. Then 20 μL of 1M Tris pH 8.0 (15568-025 UltraPure, Invitrogen, Carlsbad, Calif.) was added. Isotope-labeled peptides were added as internal standards (IS). This suspension was incubated overnight with tumbling at 4° C. to achieve peptide capture. The next day, the antibody bead:peptide complexes were washed twice with 100 μL PBS+0.01% CHAPS and once in 100 μL 0.01% PBS+0.01% CHAPS. Finally, the peptides were eluted by incubation in 30 μL of 5% acetic acid/3% ACN. Released peptides were stored at −80° C. until analysis. For samples analyzed from the WA State NBS laboratory, procedures were identical to those for previous samples except that volumes were reduced as follows: 58.1 μL of 1×PBS+0.03% CHAPS, 0.59 μg pAb for each peptide, 3.13 μL internal standard (IS), and 12.5 μL TRIS.
Liquid Chromatography-Tandem Mass Spectrometry. Enriched samples were analyzed at two laboratory sites to examine the inter-laboratory variability in data acquisition, utilizing two separate LC-MS/MS systems and instrument configurations (described below). Measured peptide concentrations were then compared for method validation. Peptide parent and daughter ion spectra have been previously reported. Kerfoot et al. Proteomics Clin Appl. 2012; 6: 394-402.
Laboratory Site 1: Instruments included a Waters Xevo TQ-XS MS with ionkey source technology connected to Waters M-Class Gradient and Loading pumps (Waters, Milford, Mass.). Chromatographic solvents were A: H₂O+0.1% Formic Acid (FA) and B: ACN+0.1% FA. Initially, peptide mixtures were loaded onto a M-Class Trap Symmetry 300 μm×50 mm C18 column (100 Å, 5 μm) utilizing a constant flow of 98:2 A:B at 20 μL/min for 3 minutes. Subsequently, the flow was reversed and peptides were separated using gradient flow across a 150 μm×100 mm BEH C18 ikey (130 Å, 1.7 μm). Gradient method programming is shown in Table 4. The peptides monitored in this location were CD3ε 197, WASp 274, WASp 289, BTK 407, and ATP7B 1056.

TABLE 4

LC method setup for signature peptide separation
at Laboratory Site 1 and Laboratory Site 2.
A = H₂O + 0.1% Formic Acid, B = B: ACN + 0.1% Formic Acid

Lab site

1	Lab site 2

Time	% B	Time	% B

0	5	0	1
1	5	4	1
11	45	24	40
13	85	25	90
15	85	26	90
17	5	27	1
20	5	35	1

Parameters for transitions and collision energy (CE) were taken from a linear regression of previously optimized values in Skyline and those generated using Waters intellistart technology to identify the most intense fragments upon ionization. SRM transitions were acquired at unit/unit resolution in both the Q1 and Q3 quadrupoles with 5 ms dwell time and 3 ms pause between mass ranges, resulting in a cycle time of 1.5 s. All samples were run in a blinded fashion.
Laboratory Site 2: LC-MS was conducted on a SCIEX 5500 QTRAP mass spectrometer interfaced with an Eksigent 425 LC and Nanoflex Chip system. Chromatographic solvents were A: H₂O+0.1% FA and B: 90% ACN+0.1% FA. Peptides were loaded on a 0.2×0.5 mm trap column (Reprosil-Pur AQ C18, 3 μm, 120 A) at 2% B using a flow rate of 4 μL/min for 4 minutes. Peptides were eluted on a 0.075×150 mm column (Reprosil-Pur AQ C18, 3 μm, 120 A) at 300 nL/min. The gradient program is shown in Table 4. Collision energy settings were taken from Skyline. MacLean et al. Bioinformatics. 2010; 26: 966-968. Transitions were acquired at unit/unit resolution with a 10 ms dwell time and 5 ms pause between mass ranges resulting in a cycle time of 0.75 sec. All data were acquired in a blinded fashion.
Method Performance Assessment. A response curve was performed to determine assay linearity and sensitivity in a background matrix of DBS. Punches from normal control DBS (4 punches per sample) were extracted using extraction buffer (ProteaseMax™, ammonium bicarbonate) in triplicate. Trypsin digestion was performed on the extracted protein, and the digests were pooled to create a common background matrix. Heavy stable isotope standards were spiked into the digest and serially diluted to create samples with varying peptide amounts (2000, 200, 12.5, 4.17, 1.39, 0.69 fmol). Two micrograms of each antibody, covalently coupled to magnetic Protein G beads, were added to the background matrix and incubated overnight. The antibody beads were washed with PBS, and the eluate was analyzed by SRM.
Repeatability and intra- and inter-assay precision were characterized by performing measurement of endogenous (light) peptide signal over 5 separate days. Each sample was analyzed in 5 complete process replicates (including punches, extraction, digestion, enrichment, and mass spectrometry) per day.
Finally, stability was assessed by comparing the endogenous (light) peptide detected in DBS stored at room temperature for 1 day and 3 days to peptide detected in DBS at −80° C. in a sealed container. Each sample was processed as described above in process triplicate. Percent difference was calculated at each timepoint.
Interlaboratory Validation of the Analytical Assay. DBS extractions, trypsin digestion, and peptide captures for patient samples were all performed at Laboratory Site 1. Peptide solutions eluted from antibody-beads were split into two 15 μl aliquots for analysis at Laboratory Site 1 and Laboratory Site 2 for inter-laboratory validation of the analytical performance of the assay.
Data Analysis. All SRM data were analyzed and plotted using Skyline (MacCoss Lab Software, open source, Seattle, Wash.). MacLean et al. Bioinformatics. 2010; 26: 966-968. Endogenous target peptide concentrations were quantified by comparing the ratio of the peak area of the signature peptide to its IS added at a known concentration (100 fmol). Statistics were generated using Graphpad Prism (San Diego, Calif.). Receiver operating characteristic (ROC) curves were constructed using Graphpad Prism and a 95% confidence interval.
Results. Peptide Selection and Antibody Development. Selected peptide sequences, molecular weights, parent, and daughter ions are listed in FIG. 1. Fragmentation patterns for the peptides of interest have been previously reported. Kerfoot et al. Proteomics Clin Appl. 2012; 6: 394-402. Affinity-purified polyclonal antibodies (Pacific Immunology, Ramona, Calif.) were generated against all five peptides and pursued for use in human samples because of their ability to successfully capture their target sequences and the absence of background signals brought on by copurified peptide contaminants. Kuhn et al. Clin. Chem. 2009; 55: 1108-1117; Hoofnagle et al. Clin. Chem. 2008; 54: 1796-1804; Whiteaker et al. Mol. Cell Proteomics. American Society for Biochemistry and Molecular Biology; 2011; 10: M110.005645.
Method Performance Assessment. Analytical figures of merit are reported in Table 5. Overall, the linear response spanned a range from 1.39 to 2000 fmol of peptide (FIGS. 2A-2E). The median coefficient of variation (CV) for all points on the response curve was 11%. Lower limits of quantification (LLOQ) were defined by the lowest point to yield a CV <20%. LLOQs ranged from 0.69 to 12.5 fmol. There were five peptides detected above LLOQ in the DBS samples. Across all peptides, the mean intra-assay (i.e. within-day) variability ranged from 11 to 22% while the inter-assay (i.e. between-day) variability ranged from 11 to 43%. Of note, a single peptide (BTK 545-558) showed variability greater than 20% CV

TABLE 5

Analytical performance of signature peptides

%

Infra-

Inter-

Relative

(fmol)

Median

assay

Difference

Protein	Peptide	LLOD	ULOD	LLOQ	CV	CV	CV	24 h	72 h

CD3ε	CD3ε 197-205	0.69	2000	0.69	13	12	11	0.17	0.09
BTK	BTK 407-417	0.69	2000	1.39	10	14	25	0.32	−0.05
	BTK 545-558	0.69	2000	1.39	12	22	43	0.61	0.06
WASp	WASp 274-288	0.69	2000	0.69	17	11	12	−0.04	0.01
	WASp 289-304	1.39	2000	4.17	7	13	17	−0.19	0.03

Finally, stability was assessed by comparing the endogenous (light) peptide detected in DBS stored at room temperature for 1 day and 3 days to peptide detected in DBS preserved at −80° C. in a sealed container. Results are reported in Table 5. All five peptides had endogenous signal above the LLOQ and little variability over time. Representative multiple reaction monitoring (MRM) chromatograms for each peptide are shown in FIGS. 3A-3E.
Overall, there was high level of agreement between the concentrations determined by two separate instrumental analyses. Correlation plots comparing the two measurements, FIGS. 4A-4D, show the linearity of measured concentrations with R²values 0.97 in the cases of primary peptides CD3ε 197 (FIG. 4B), WASp 274 (FIG. 4A), BTK 407 (FIG. 4C), and ATP7B 1056 (FIG. 4D). The measurements of WASp 289 was found to correlate with R²=0.85 and could therefore be useful as a secondary marker to WASp 274 (FIG. 5).
Peptide Concentrations. After analysis, normal controls were unblinded to define normal ranges for affected patient comparison. The average peptide concentrations from normal controls were as follows (average ±SD): CD3ε=228.68±150.98 pmol/L, WASp 274=1176.96±456.68 pmol/L, WASp 289=10326.98±4513.13 pmol/L, BTK 407=635.09±260.40 pmol/L, and BTK 545=1038.44±465.77 pmol/L. Analysis of signature peptides found statistically significant (p<0.05-0.0001) reductions in patient peptide levels relative to control groups in each case (FIGS. 6A-6E). Peptide levels in the majority of affected patients were significantly diminished or absent. For each patient, the concentration of ATP7B 1056 was also determined using previously developed immuno-SRM methodology. Jung et al. 2017, supra. These protein concentrations serve as quality control (QC) measurements and their consistency across samples is used to assess digestion and process reproducibility (FIGS. 3A-3E).
Peptide concentration cutoffs for each PIDD diagnosis were arbitrarily set at −1.25 SD (CD3ε), −2.15 SD (WASp 274), −1.75 SD (WASp 289), −2 SD (BTK 545), and −2.25 SD (BTK 407). Use of these ranges resulted in 2 false positive indications in the normal controls. NC4 and NC20 were indicated to be WAS and SCID patients respectively. NC signature peptide values are shown in FIG. 8. Cutoffs for positive identification of PIDDs are shown in Table 6.

TABLE 6

Cutoffs for signature peptides by concentration

BTK

545	BTK 407	WASp 274	WASp 289	CD3ε 197
	(pmol/L)	(pmol/L)	(pmol/L)	(pmol/L)	(pmol/L)

Average	1038.44	635.09	1176.96	10326.98	228.68
SD	465.77	260.40	456.68	4513.13	150.98
Cutoff	106.90	49.19	195.10	2428.99	39.96

Using these cutoffs, the specific PIDD diagnosis was predicted for each patient. Predicted diagnoses showed excellent agreement with clinical or genetic diagnoses as shown in FIG. 9. Every molecularly-confirmed case of WAS and BTK was also diagnosed by immuno-SRM analysis. Two patients, Patient 10 and 13, who were clinically diagnosed as agammaglobulinemia, had normal levels of BTK protein by immuno-SRM. Molecularly, no mutations in BTK were identified in these patients. Segundo et al. Front Immunol. Frontiers; 2018; 9: 289. Interestingly, patient 12 with agammaglobulinemia had low levels of BTK protein but no mutations were found in the coding regions of BTK. In addition, one case of X-linked hypomorphic SCID, patient 41, was identified as normal by immuno-SRM. For each signature peptide utilized, area under the curve (AUC) analysis of the ROC plots reveal areas from 0.925 to 0.999 with p-values ranging from 0.015-0.0001 (FIGS. 10A, 10B). Overall 97.6% of cases had concordance between the clinical diagnosis and the immuno-SRM assay results. Interesting outlier and discordant cases are discussed further below.
Signature peptide concentrations for NBS lab samples are shown in FIG. 11. Each DBS sample had significant measured peptide concentrations above the previously set diagnostic cutoffs for PIDDs, indicating unaffected status.
Immuno-SRM as a sensitive and specific proteomic screening method for the multiplex detection of patients with three life-threatening PIDD (i.e., SCID, WAS, and XLA) from DBS has been demonstrated. The results clearly differentiate patients with PIDD from normal controls, with low levels of endogenous peptides of transmembrane protein CD3ε and intracellular proteins WASp and BTK correlating with the target diseases (SCID, WAS, and XLA, respectively). These diagnoses can be made in a single run with a total runtime of 20 min or 6.67 min per disease target. The disclosed results also demonstrate peptide stability in DBS, with minimal variability in concentrations after 72 hours of storage at room temperature (Table 5).
The immuno-SRM platform reliably detected endogenous peptide from normal control
DBS in this highly multiplexed fashion. Normal control DBS (N=40) were unblinded and utilized to define the normal ranges and potential screen-positive cutoffs (FIG. 8). In clinical laboratories, reference ranges for diagnostic tests are determined by the normal distribution in the general population. Initial cutoffs for screening tests are typically conservative, aiming to detect all true positives without creating an excessively high screen positive rate relative to the incidence of disease (Table 6). However, these cutoffs are continually validated and adjusted in accordance with population-based studies. Given these parameters, the definition of screen-positive results ranged from 1.25-2.25 standard deviation (SD) below the mean for the peptides in this example. The chosen cutoffs generated 2 false positive normal controls, one WAS (NC4) and one SCID (NC20) (FIG. 8). In the case of NC4, rescreening showed WASp levels in the normal range. These preliminary cutoffs are not static and will become better defined as higher numbers of normal controls and patient samples are screened.
Using these cutoffs, every molecularly-confirmed WAS and BTK patient covering a broad range of mutations were positively identified (FIG. 9). As hypothesized, peptide concentrations are reduced in the majority of BTK and WAS cases, independent of genotype. Qasim et al. Br. J. Haematol. 2001; 113: 861-865; Jin et al. Blood. American Society of Hematology; 2004; 104: 4010-4019; Futatani et al. British Journal of Haematology. 2001; 114(1): 141-9. These peptides therefore provide biomarkers for diagnosis and screening. Of the 3 SCID patients available for testing, 2 were positively identified by CD3ε analysis. The third patient, while having low CD3ε levels relative to the majority of the normal controls, was within the defined cutoffs and had a “hypomorphic” mutation in IL2RG known to generate a partially functional protein. This is reflected by the patients total CD3+ T-cell count that was mildly low (800 cells/μL) but not absent as in the classical form of SCID. Since CD3ε is exclusively expressed by CD3+ T cells in peripheral blood, the amount of CD3ε protein present is reflective of total CD3+ T cell counts. Therefore, patients with hypomorphic forms of SCID, patients with “leaky” forms of SCID who have expanded oligoclonal T cell populations, or patients who have expanded maternally-derived T cells, may be missed by the Immuno-SRM approach.
ROC curves were constructed to assess the diagnostic ability of immuno-SRM analysis. These plots relate the true positive rate to the false positive rate with increasingly stringent cutoff values. As diagnostic cutoffs are lowered, the test will have greater ability to note true positives, but this process is also more likely to lead to false positives. A screening test maintaining a high true positive rate and a low false positive rate will therefore lead to graphs lying close to the y axis and a large AUC (FIGS. 10A, 10B). These values indicate high diagnostic accuracy for immuno-SRM analysis of signature peptides of PIDDs.
QC monitoring of digestion and process performance is included in the current immuno-SRM multiplex in the form of ATP7B signature peptide measurements. As not all detected metabolites are helpful NBS targets, the calculation of metabolite ratios and secondary metabolite analysis are employed to improve the sensitivity and specificity of NBS for certain diseases, such as the C3:C2 ratio and 2-methylcitric acid analysis in methylmalonic aciduria. Lindner et al. J. Inherit. Metab. Dis. 2nd ed. 2008; 31: 379-385. In addition, target ratioing can account for variability between samples brought on by a number of factors including sample collection quality, storage, extraction and digestion efficiency, and blood characteristics. Razavi et al. Bioanalysis. Future Science Ltd London, UK; 2016; 8: 1597-1609. Here, ATP7B concentrations were found to be largely consistent across the screened samples (FIG. 7). Absent ATP7B could serve to flag improperly processed or handled specimens. As an initial experiment, each PIDD peptide was compared by ratio to the endogenous concentration of ATP7B in the same sample. The resulting predictions based on peptide concentrations showed complete agreement with the clinical diagnosis, demonstrating immuno-SRM and ATP7B ratioing is an effective and complementary tool for PIDD diagnosis (FIG. 12 and Table 7). These types of ratios have utility in clinical immuno-SRM screening, provided the chosen peptide is proven to be a ubiquitous and significantly invariant signal across a large cohort of samples.

TABLE 7

Cutoffs for Signature Peptides by the Ratios against ATP7B peptide

BTK

545	BTK 407	WASp 274	WASp 289	CD3ε 197
	ATP7B	ATP7B	ATP7B	ATP7B	ATP7B
	Ratio	Ratio	Ratio	Ratio	Ratio

Average	9.31	5.71	10.71	92.47	2.12
SD	4.62	2.68	5.10	48.79	1.55
Cutoff	1.23	1.16	2.04	24.16	0.33

One case demonstrated the importance of having both primary and secondary signature peptides for proteins of interest. Patient 18 was found to have a positive BTK diagnosis after the analysis of BTK 545 instead of primary marker BTK 407. Levels of BTK 407 were significantly reduced relative to the average, 167.86 versus 642.16 pmol/L, but not quite low enough to trigger a positive screen. In contrast, BTK 545 levels were nearly absent (FIG. 9) because the patient harbors the p.Y551N mutation, which is located within the amino acid sequence 545-558 encompassed by the signature peptide itself. In this case, the multiplexed peptides allowed for confirmation of a positive diagnosis that was initially borderline.
It was notable that normal levels of BTK were found in two clinically defined agammaglobulinemic patients (sample #10 and #13) who lacked mutations in BTK by Sanger sequencing (FIG. 9). These patients therefore likely do not have XLA but may have other autosomal forms of agammaglobulinemia, although broader genetic testing was not performed. Another patient (sample #12) had diminished levels of BTK protein but no identifiable mutation in BTK. This suggests the mutation may have been missed during sequencing of the coding region and intron-exon junctions or the patient may harbor a BTK mutation affecting either the regulatory elements, Poly-adenylation signal, or intronic regions. These cases highlight the clinical utility of immuno-SRM.
Additionally, two samples obtained from the same WAS patient pre- and post-bone marrow transplant (BMT) ( samples # 29 and 30 in FIG. 9, respectively). Pre-BMT, immuno-SRM analysis identified the patient as having WAS. Post-BMT, the patient was identified as normal. This case highlights the ability of immuno-SRM to follow the therapeutic course of BMT and confirm successful reconstitution of the immune system. A similar principle can be applied to patients with monogenetic disorders undergoing gene therapy.
Overall, the analysis demonstrates that the disclosed assay has a broad linear range and acceptable precision to determine the concentrations of target peptides in DBS (Table 5). Correlation plots show significant concordance of sample analysis by different MS instruments in two separate laboratory facilities (FIGS. 4A-4D and 5). Four of the five peptides, CD3ε 197, WASp 274, BTK 407, and ATP7B 1056 were nearly identical upon analysis with R²values >0.97. WASp 289 showed slightly more variable performance with an overlap of R²=0.85 and would therefore likely be a secondary marker to WASp 274 when conducting clinical analysis. Additionally, BTK 545 showed a variability greater than 20% CV, which would make it suitable as a secondary marker to BTK 407. These results show that immuno-SRM analysis has clinical application and transferability. In a clinical setting, the use of reference standards and/or calibrators will aid inter-laboratory validation of findings.
Randomly selected samples provided by the NBS laboratory of Washington State were used to test the feasibility of utilizing immuno-SRM analysis in the context of NBS. Due to limited sample availability and to test the utility of signature peptide analysis from a smaller sample, the amount of DBS used was reduced from 1 whole spot to 5 or 6 3-mm punches. Peptides of interest were readily enriched and analyzed with minimal change to sample processing. The concentrations of signature peptides were all greater than the pre-defined cutoffs obtained from analysis of known normal controls (FIG. 11). These patients would therefore be designated as normal. The ability to robustly perform this analysis with a greatly reduced sample input makes immuno-SRM analysis more amenable to translation into NBS. This high-throughput multiplexed method may effectively decrease run time per disease, making it suitable for NBS where current automated methods have a typical run time of less than three minutes. Rashed et al. Clin. Chem. 1997; 43: 1129-1141; Khalid et al. J Med Screen. SAGE Publications, Sage UK: London, England; 2008; 15:112-117. The successful prediction of BTK patients using DBS shipped at ambient temperature via traditional post from Vietnam also highlights the potential utility for diagnostic testing in resource poor settings where collection and shipping of DBS is economical.
NBS has been one of the most successful public health initiatives in modern times but relies on the detection of accumulated metabolites due to downstream enzyme deficiency. However, many genetic disorders including PIDD are characterized by absent or decreased proteins, limiting the scope of current NBS methods. Qasim et al. Br. J. Haematol. 2001; 113: 861-865; Jin et al. Blood. American Society of Hematology; 2004; 104: 4010-4019. By being able to detect PIDD-related peptides from DBS, immuno-SRM bridges this gap in current coverage, allowing for the expansion of NBS to treatable diseases currently without metabolite biomarkers. Immuno-SRM would rapidly provide quantified evidence of protein deficiency and could be performed simultaneously with initial screening and molecular analysis from DBS without further invasive procedures. Quantification of these signature peptides lays the foundation for immuno-SRM as a highly multiplexable screening and diagnostic tool for various congenital diseases.
Example 2. Development of a quantitative assay for NBS of cystinosis using DBS. An immuno-SRM assay was performed as described in Example 1 but using signature peptides for cystinosis. Antibodies recognizing CTNS 115 peptide were developed and used for screening in patient samples. This peptide biomarker provides robust and specific detection of CTNS down to 16.9 pmol/L in DBS (Table 8). Measured concentrations for CTNS range from 5.5-79.8 pmol/L. Levels of SHPK range from 922.3-5787.6 pmol/L. Samples CTNS 00001-CTNS 00013 are blinded and predicted mutational status is based on protein levels evidenced by immuno-SRM. The most common form of cystinosis results from a 57 kb-deletion mutation that results in the loss of CTNS protein as well as the adjacent protein SHPK. Samples in bold are either confirmed or predicted to contain homozygous 57-kb deletion mutations. ND: Not Detected (Table 8).

TABLE 8

Concentrations of CTNS and SHPK proteins in patients and normal control samples.

	pmol/L
Samples	CTNS	pmol/L SHPK	Status	Mutation

CTNS

209	ND	1026.193	Confirmed	57kb del/ c.838A > G;
				p.K280R
CTNS
211	ND	3577.042	Confirmed	c. DNA 18656 C < G/c. DNA
				21635 C < T
CTNS
212	ND	4563.733	Confirmed	c.473T > C; p.L158P/
				c.530A > G; p. N177S
CTNS
213	ND	3717.015	Confirmed	c.473T > C; p.L158P/
				c.530A > G; p. N177S
CTNS
243	ND	ND	Confirmed	57 kb del/57 kb del
CTNS 252	ND	ND	Confirmed	57 kb del/57 kb del
CTNS 00001	ND	1426.654	Blinded
CTNS 00002	ND	3941.397	Blinded
CTNS 00003	ND	2736.816	Blinded
CTNS 00004	ND	5787.633	Blinded
CTNS 00005	ND	ND	Blinded	Predicted - 57 kb del/57 kb
				del
CTNS 00006	ND	991.878	Blinded
CTNS 00007	ND	922.330	Blinded
CTNS 00008	71.87953553	469.439	Blinded	Predicted - Normal Control
CTNS 00009	ND	ND	Blinded	Predicted - 57 kb del/57 kb
				del
CTNS 0010	ND	ND	Blinded	Predicted - 57 kb del/57 kb
				del
CTNS 00011	ND	ND	Blinded	Predicted - 57 kb del/57 kb
				del
CTNS 00012	ND	1087.535	Blinded
CTNS 00013	79.79928237	3401.707	Blinded	Predicted - Normal Control
Normal Controls
NM1	46.23052899	1177.935	Confirmed
NM2	ND	2000.437	Confirmed
NM3	46.63169607	1226.747	Confirmed
NF1	ND	1784.461	Confirmed

Example 3. Multiplex immuno-SRM assay of CTNS and SHPK peptides for diagnosis of cystinosis. Multiplexed quantification of peptide biomarkers for CTNS and SHPK from affected and normal control DBS has been achieved in a single experiment, and the results are consistent with underlying patients' genotypes. CTNS 115 peptide was joined with a signature peptide biomarker for sedoheptulokinase (SHPK), SHPK 363, for a multiplexed analysis of cystinosis patient samples. These assays involved the combined use of antibodies for both CTNS 115 and SHPK 363 in the immuno-SRM workflow. Four micrograms of CTNS 115 mAb and 0.25 micrograms of SHPK 363 mAb were used in this analysis. The multiplexed analysis was performed on 4 normal controls, 6 confirmed cystinosis patients, and 13 blinded samples (Table 8). CTNS 115 is completely absent from the known patient samples. In addition, patient samples confirmed to be homozygous for the 57-kb deletion lack SHPK 363 while heterozygous patient samples have varying levels of SHPK protein present. Interestingly, sample CTNS 209 (compound heterozygous for the 57 kb del and c.838A>G; p.K280R) has reduced levels of SHPK relative to samples CTNS 211-213, where no deletion is present (FIG. 13), suggesting correlation between genotype and peptide levels. Of the 13 blinded samples, only two have detectable concentrations of CTNS. These samples are predicted to be normal controls. The assay also accurately predicted that samples 00005 and 00009-00010 were from patients with homozygous deletions as they were lacking in both CTNS and SHPK peptides (Table 8).
Based on an initial multiplexed quantification of CTNS 115 and SHPK 363, it was determined that enhanced sensitivity was necessary to increase CTNS 115 signal. This prompted a transition from microliter per minute flow rates during LC-MS/MS detection using a Waters lonkey system to a more sensitive nanoliter per minute LC-MS/MS assay. This change has greatly enhanced the analytical sensitivity of the measurement. Direct comparison of CTNS 115 when analyzed on both systems finds that nano-flow analysis boosts peptide signal-to-noise (S/N) ratios by 44-fold at 123 amol, 16-fold at 3 amol and 5-fold at 0.3 amol (FIG. 14). This lowers the limit of quantification for CTNS 115 in DBS significantly. In addition, the increased signal was obtained using ½ of the sample typically injected for LC-MS/MS analysis. This allows for a reduction in the amount of DBS sample necessary for future screening.
With this improved assay, 9 blinded samples and 4 normal controls were screened (FIG. 15). Seven of the nine samples were found to be lacking CTNS 115 entirely. Six of those seven were additionally lacking SHPK 363 and were predicted to harbor the 57-kb homozygous deletion mutation which would impair the production of both proteins. The remaining three patients were predicted to be heterozygotes. Two of the predicted heterozygotes were found to have levels of CTNS 115 that were moderately to significantly reduced relative to controls. These predictions were confirmed in six cases, including the three compound heterozygotes. The sequencing of the remaining three patients is ongoing but are predicted to be 57-kb deletion homozygotes by immuno-SRM. Normal controls were found to have appreciable levels of both CTNS 115 and SHPK 363.
Incorporation of validated peptide biomarkers from other proteins will allow for the development of robust clinical reference ranges for process monitoring and analytical quality control downstream. Antibodies for WD (ATP7B protein) and primary immunodeficiency disorders (BTK and WAS proteins) are being incorporated. These ranges will inform the clinical validity of any sample run in a population screening environment, while simultaneously serving as diagnostic screens of their own associated disease states.
Example 4. Additional CTNS and SHPK peptide biomarkers are being developed. Peptides CTNS 120, CTNS 194, SHPK 44, and SHPK 388 showed robust immune response and high polyclonal antibody titer generation. Therefore, anti-CTNS 120, anti-CTNS 194, anti-SHPK 44, and/or anti-SHPK 388 antibodies may be used in the disclosed methods to screen for cystinosis. Polyclonal antibodies for SHPK 388 have been utilized previously to show the presence of this peptide in normal control DBS and its absence in patients' DBS. Monoclonal antibodies for the CTNS 120, CTNS 194, SHPK 44, and SHPK 388 peptide biomarkers will be produced. These markers will be incorporated into existing multiplexed assays.
Example 5. Polyclonal antibodies or monoclonal antibodies were used to enrich for WASp 274 or BTK 407 peptides. Internal standard peptide peak areas were generated upon peptide capture of both WASp 274 (FIGS. 16B and 16D) and BTK 407 (FIGS. 16A and 16C). Peak areas were measured in blank control samples only (i.e. no patient blood, FIGS. 16C, 16D) and in an aggregated set of both patient and blank control samples (FIGS. 16A and 16B). Monoclonal antibodies showed comparable or superior ability to capture and enrich target peptides regardless of sample matrix.
Example 6. Extension of cystinosis screen. The screen for cystinosis using signature peptide biomarkers CTNS 115 and SHPK 363 as described in Examples 2 and 3 (FIG. 13 and Table 8) was extended to include 90 patients in total. After analysis, cystinosis patients were unblinded to find that CTNS levels were markedly reduced relative to control. The vast majority (87/90, 96.7%) have levels of CTNS 115 below the diagnostic cutoff of 1.5 standard deviations below the normal mean regardless of mutation (FIG. 17). In addition, cystinosis patient mutations could be stratified based on SHPK concentration. Of patients with a known homozygous 57-kb deletion genetic background, 26/27 (96.3%) had non-detectable levels of SHPK 363. These patients fell below a diagnostic cutoff set at 1.8 standard deviations below the normal mean. In addition, compound heterozygous patients with a single 57-kb deletion mutation were found to have significantly lower SHPK 363 levels than those patients with no copies of this deletion mutation. As expected, these patients successfully produced SHPK in proportion to their genotype (FIG. 17).
Example 7. A study on WD patients. An immuno-SRM assay was performed as described in Example 1 but using DBS samples from normal controls and 16 WD patients and monoclonal antibodies binding to the following signature peptides for WD: ATP7B 1056, ATP7B 214, and ATP7B 887 (FIGS. 18A-180). In this study, all 16 WD patients showed reduced or absent level of ATP7B 1056 (less than 2.6 SD below the average of normal control; FIG. 18A). Both ATP7B 214 and ATP7B 887 peptide levels were reduced as well compared to the normal controls (FIGS. 18B, 18C). In each case, every WD patient was identified as having levels of the ATP7B peptide biomarkers that are below that of normal controls. Each has potential utility as an NBS screening tool and together they provide a robust complimentary confirmation of WD status.
As will be understood by one of ordinary skill in the art, each embodiment disclosed herein can comprise, consist essentially of or consist of its particular stated element, step, ingredient or component. Thus, the terms “include” or “including” should be interpreted to recite: “comprise, consist of, or consist essentially of.” The transition term “comprise” or “comprises” means includes, but is not limited to, and allows for the inclusion of unspecified elements, steps, ingredients, or components, even in major amounts. The transitional phrase “consisting of” excludes any element, step, ingredient or component not specified. The transition phrase “consisting essentially of” limits the scope of the embodiment to the specified elements, steps, ingredients or components and to those that do not materially affect the embodiment. A material effect would cause a statistically significant reduction in the ability to reliably diagnose SCID, WAS, XLA, cystinosis, or Wilson Disease utilizing DBS obtained from a newborn, the antibodies disclosed herein, and immuno-SRM.
Unless otherwise indicated, all numbers expressing quantities of ingredients, properties such as molecular weight, reaction conditions, and so forth used in the specification and claims are to be understood as being modified in all instances by the term “about.” Accordingly, unless indicated to the contrary, the numerical parameters set forth in the specification and attached claims are approximations that may vary depending upon the desired properties sought to be obtained by the present invention. At the very least, and not as an attempt to limit the application of the doctrine of equivalents to the scope of the claims, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques. When further clarity is required, the term “about” has the meaning reasonably ascribed to it by a person skilled in the art when used in conjunction with a stated numerical value or range, i.e. denoting somewhat more or somewhat less than the stated value or range, to within a range of ±20% of the stated value; ±19% of the stated value; ±18% of the stated value; ±17% of the stated value; ±16% of the stated value; ±15% of the stated value; ±14% of the stated value; ±13% of the stated value; ±12% of the stated value; ±11% of the stated value; ±10% of the stated value; ±9% of the stated value; ±8% of the stated value; ±7% of the stated value; ±6% of the stated value; ±5% of the stated value; ±4% of the stated value; ±3% of the stated value; ±2% of the stated value; or ±1% of the stated value.
Notwithstanding that the numerical ranges and parameters setting forth the broad scope of the invention are approximations, the numerical values set forth in the specific examples are reported as precisely as possible. Any numerical value, however, inherently contains certain errors necessarily resulting from the standard deviation found in their respective testing measurements.
The terms “a,” “an,” “the” and similar referents used in the context of describing the invention (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. Recitation of ranges of values herein is merely intended to serve as a shorthand method of referring individually to each separate value falling within the range. Unless otherwise indicated herein, each individual value is incorporated into the specification as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., “such as”) provided herein is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention otherwise claimed. No language in the specification should be construed as indicating any non-claimed element essential to the practice of the invention.
Groupings of alternative elements or embodiments of the invention disclosed herein are not to be construed as limitations. Each group member may be referred to and claimed individually or in any combination with other members of the group or other elements found herein. It is anticipated that one or more members of a group may be included in, or deleted from, a group for reasons of convenience and/or patentability. When any such inclusion or deletion occurs, the specification is deemed to contain the group as modified thus fulfilling the written description of all Markush groups used in the appended claims.
Certain embodiments of this invention are described herein, including the best mode known to the inventors for carrying out the invention. Of course, variations on these described embodiments will become apparent to those of ordinary skill in the art upon reading the foregoing description. The inventor expects skilled artisans to employ such variations as appropriate, and the inventors intend for the invention to be practiced otherwise than specifically described herein. Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context.
Furthermore, numerous references have been made to patents, printed publications, journal articles and other written text throughout this specification (referenced materials herein). Each of the referenced materials are individually incorporated herein by reference in their entirety for their referenced teaching.
In closing, it is to be understood that the embodiments of the invention disclosed herein are illustrative of the principles of the present invention. Other modifications that may be employed are within the scope of the invention. Thus, by way of example, but not of limitation, alternative configurations of the present invention may be utilized in accordance with the teachings herein. Accordingly, the present invention is not limited to that precisely as shown and described.
The particulars shown herein are by way of example and for purposes of illustrative discussion of the preferred embodiments of the present invention only and are presented in the cause of providing what is believed to be the most useful and readily understood description of the principles and conceptual aspects of various embodiments of the invention. In this regard, no attempt is made to show structural details of the invention in more detail than is necessary for the fundamental understanding of the invention, the description taken with the drawings and/or examples making apparent to those skilled in the art how the several forms of the invention may be embodied in practice.
Definitions and explanations used in the present disclosure are meant and intended to be controlling in any future construction unless clearly and unambiguously modified in the following examples or when application of the meaning renders any construction meaningless or essentially meaningless. In cases where the construction of the term would render it meaningless or essentially meaningless, the definition should be taken from Webster's Dictionary, 3rd Edition or a dictionary known to those of ordinary skill in the art, such as the Oxford Dictionary of Biochemistry and Molecular Biology (Eds. Attwood T et al., Oxford University Press, Oxford, 2006).

Claims

1. A method of screening for severe combined immunodeficiency (SCID), Wiskott-Aldrich syndrome (WAS), and/or X-linked agammaglobulinemia (XLA) in a subject, the method comprising:

Obtaining a dried blood spot (DBS) sample derived from the subject;

Digesting proteins from blood of the DBS with an enzyme to yield one or more peptides;

Enriching for:

a CD3ε signature peptide of SCID of SEQ ID NO: 1 with an antibody or antigen-binding fragment thereof that binds the CD3ε signature peptide and comprises: a heavy chain variable (VH) domain comprising CDR1 of SEQ ID NO: 22, CDR2 of SEQ ID NO: 23, and CDR3 of SEQ ID NO: 24, and a light chain variable (VL) domain comprising: CDR1 of SEQ ID NO: 25, CDR2 of SEQ ID NO: 26, and CDR3 of SEQ ID NO: 27;

a first WASp signature peptide of WAS of SEQ ID NO: 2 with an antibody or antigen-binding fragment thereof that binds the first WASp signature peptide and comprises: a heavy chain variable (VH) domain comprising CDR1 of SEQ ID NO: 28, CDR2 of SEQ ID NO: 29, and CDR3 of SEQ ID NO: 30, and a light chain variable (VL) domain comprising: CDR1 of SEQ ID NO: 31, CDR2 of SEQ ID NO: 32, and CDR3 of SEQ ID NO: 33;

a second WASp signature peptide of WAS of SEQ ID NO: 3 with an antibody or antigen-binding fragment thereof that binds the second WASp signature peptide;

a first BTK signature peptide of XLA of SEQ ID NO: 4 with an antibody or antigen-binding fragment thereof that binds the first BTK signature peptide and comprises: a heavy chain variable (VH) domain comprising CDR1 of SEQ ID NO: 34, CDR2 of SEQ ID NO: 35, and CDR3 of sequence GDI, and a light chain variable (VL) domain comprising: CDR1 of SEQ ID NO: 36, CDR2 of SEQ ID NO: 37, and CDR3 of SEQ ID NO: 38; and/or

a second BTK signature peptide of XLA of SEQ ID NO: 5 with an antibody or antigen-binding fragment thereof that binds the second BTK signature peptide;

Performing liquid chromatography-multiple reaction monitoring mass spectrometry (LC-MRM-MS) on the enriched peptides to determine concentrations of the peptides; and

Diagnosing the subject with:

SCID when the concentration of the CD3ε signature peptide is lower than a predetermined threshold concentration or when the CD3ε signature peptide is absent;

WAS when the concentrations of the first and/or second WASp signature peptides are lower than corresponding predetermined threshold concentrations or when the first and/or second WASp signature peptides are absent; and

XLA when the concentrations of the first and/or second BTK signature peptides are lower than corresponding predetermined threshold concentrations or when the first and/or second BTK signature peptides are absent.

2. The method of claim 1, wherein the method is performed as part of a newborn screening (NBS) that additionally screens the subject for one or more of phenylketonuria, primary congenital hypothyroidism, cystic fibrosis, and sickle cell disease.

3. The method of claim 1, wherein the method is performed in the absence of clinical symptoms of SCID, WAS, and/or XLA in the subject.

4. The method of claim 1, wherein the enzyme is trypsin.

5. The method of claim 1, wherein the corresponding predetermined threshold concentration for each signature peptide is calculated from a standard deviation of the mean concentration of each signature peptide in DBS from a population of normal control subjects.

6. The method of claim 5, wherein the mean concentration of the CD3ε signature peptide of SCID of SEQ ID NO: 1 in DBS from a population of normal control subjects comprises a concentration in a range of 70 pmol/L to 400 pmol/L.

7. The method of claim 5, wherein the mean concentration of the first WASp signature peptide of WAS of SEQ ID NO: 2 in DBS from a population of normal control subjects comprises a concentration in a range of 600 pmol/L to 5000 pmol/L.

8. The method of claim 5, wherein the mean concentration of the second WASp signature peptide of WAS of SEQ ID NO: 3 in DBS from a population of normal control subjects comprises a concentration in a range of 5500 pmol/L to 15000 pmol/L.

9. The method of claim 5, wherein the mean concentration of the first BTK signature peptide of XLA of SEQ ID NO: 4 in DBS from a population of normal control subjects comprises a concentration in a range of 350 pmol/L to 2500 pmol/L.

10. The method of claim 5, wherein the mean concentration of the second BTK signature peptide of XLA of SEQ ID NO: 5 in DBS from a population of normal control subjects comprises a concentration in a range of 550 pmol/L to 1600 pmol/L.

11. The method of claim 1, wherein the antibody or antigen-binding fragment thereof used for enrichment of the CD3ε signature peptide of SCID of SEQ ID NO: 1 comprises a VH domain of SEQ ID NO: 63.

12. The method of claim 1, wherein the antibody or antigen-binding fragment thereof used for enrichment of the CD3ε signature peptide of SCID of SEQ ID NO: 1 comprises a VL domain of SEQ ID NO: 64.

13. The method of claim 1, wherein the antibody or antigen-binding fragment thereof used for enrichment of the first WASp signature peptide of WAS of SEQ ID NO: 2 comprises a VH domain of SEQ ID NO: 65.

14. The method of claim 1, wherein the antibody or antigen-binding fragment thereof used for enrichment of the first WASp signature peptide of WAS of SEQ ID NO: 2 comprises a VL domain of SEQ ID NO: 66.

15. The method of claim 1, wherein the antibody or antigen-binding fragment thereof used for enrichment of the first WASp signature peptide of WAS of SEQ ID NO: 2 comprises a heavy chain of SEQ ID NO: 86.

16. The method of claim 1, wherein the antibody or antigen-binding fragment thereof used for enrichment of the first WASp signature peptide of WAS of SEQ ID NO: 2 comprises a light chain of SEQ ID NO: 91.

17. The method of claim 1, wherein the antibody or antigen-binding fragment thereof used for enrichment of the first BTK signature peptide of XLA of SEQ ID NO: 4 comprises a VH domain of SEQ ID NO: 67.

18. The method of claim 1, wherein the antibody or antigen-binding fragment thereof used for enrichment of the first BTK signature peptide of XLA of SEQ ID NO: 4 comprises a VL domain of SEQ ID NO: 68.

19. The method of claim 1, wherein the antibody or antigen-binding fragment thereof used for enrichment of the first BTK signature peptide of XLA of SEQ ID NO: 4 comprises a heavy chain of SEQ ID NO: 96.

20. The method of claim 1, wherein the antibody or antigen-binding fragment thereof used for enrichment of the first BTK signature peptide of XLA of SEQ ID NO: 4 comprises a light chain of SEQ ID NO: 101.

21. The method of claim 1, wherein the method further comprises screening for cystinosis by enriching for:

a first CTNS signature peptide of cystinosis of SEQ ID NO: 6 with an antibody or antigen-binding fragment thereof that binds the first CTNS signature peptide and comprises: a heavy chain variable (VH) domain comprising CDR1 of SEQ ID NO: 39, CDR2 of SEQ ID NO: 40, and CDR3 of SEQ ID NO: 41, and a light chain variable (VL) domain comprising: CDR1 of SEQ ID NO: 42, CDR2 of SEQ ID NO: 43, and CDR3 of SEQ ID NO: 44;

a second CTNS signature peptide of cystinosis of SEQ ID NO: 7 or 8 with an antibody or antigen-binding fragment thereof that binds the second CTNS signature peptide and comprises: a heavy chain variable (VH) domain comprising CDR1 of SEQ ID NO: 45, CDR2 of SEQ ID NO: 46, and CDR3 of SEQ ID NO: 47, and a light chain variable (VL) domain comprising: CDR1 of SEQ ID NO: 48, CDR2 of SEQ ID NO: 49, and CDR3 of SEQ ID NO: 50;

a third CTNS signature peptide of cystinosis of SEQ ID NO: 12 with an antibody or antigen-binding fragment thereof that binds the third CTNS signature peptide;

a fourth CTNS signature peptide of cystinosis of SEQ ID NO: 13 with an antibody or antigen-binding fragment thereof that binds the fourth CTNS signature peptide;

a first SHPK signature peptide of cystinosis of SEQ ID NO: 9 with an antibody or antigen-binding fragment thereof that binds the first SHPK signature peptide and comprises: a heavy chain variable (VH) domain comprising CDR1 of SEQ ID NO: 51, CDR2 of SEQ ID NO: 52, and CDR3 of SEQ ID NO: 53, and a light chain variable (VL) domain comprising: CDR1 of SEQ ID NO: 54, CDR2 of SEQ ID NO: 55, and CDR3 of SEQ ID NO: 56;

a second SHPK signature peptide of cystinosis of SEQ ID NO: 14 with an antibody or antigen-binding fragment thereof that binds the second SHPK signature peptide;

a third SHPK signature peptide of cystinosis of SEQ ID NO: 15 with an antibody or antigen-binding fragment thereof that binds the third SHPK signature peptide;

and

Diagnosing the subject with cystinosis when the concentrations of the first CTNS, the second CTNS, the third CTNS, the fourth CTNS, the first SHPK, the second SHPK, and/or the third SHPK signature peptides are lower than corresponding predetermined threshold concentrations or when the first CTNS, the second CTNS, the third CTNS, the fourth CTNS, the first SHPK, the second SHPK, and/or the third SHPK signature peptides are absent.

22. The method of claim 21, wherein the corresponding predetermined threshold concentration for each signature peptide is calculated from a standard deviation of the mean concentration of each signature peptide in DBS from a population of normal control subjects.

23. The method of claim 22, wherein the mean concentration of the first CTNS signature peptide of cystinosis of SEQ ID NO: 6 in DBS from a population of normal control subjects comprises a concentration in a range of 40 pmol/L to 250 pmol/L.

24. The method of claim 22, wherein the mean concentration of the first SHPK signature peptide of cystinosis of SEQ ID NO: 9 in DBS from a population of normal control subjects comprises a concentration in a range of 100 pmol/L to 8000 pmol/L.

25. The method of claim 21, wherein the antibody or antigen-binding fragment thereof used for enrichment of the first CTNS signature peptide of cystinosis of SEQ ID NO: 6 comprises a VH domain of SEQ ID NO: 69.

26. The method of claim 21, wherein the antibody or antigen-binding fragment thereof used for enrichment of the first CTNS signature peptide of cystinosis of SEQ ID NO: 6 comprises a VL domain of SEQ ID NO: 70.

27. The method of claim 21, wherein the antibody or antigen-binding fragment thereof used for enrichment of the first CTNS signature peptide of cystinosis of SEQ ID NO: 6 comprises a heavy chain of SEQ ID NO: 106.

28. The method of claim 21, wherein the antibody or antigen-binding fragment thereof used for enrichment of the first CTNS signature peptide of cystinosis of SEQ ID NO: 6 comprises a light chain of SEQ ID NO: 111.

29. The method of claim 21, wherein the antibody or antigen-binding fragment thereof used for enrichment of the second CTNS signature peptide of cystinosis of SEQ ID NO: 7 or 8 comprises a VH domain of SEQ ID NO: 71.

30. The method of claim 21, wherein the antibody or antigen-binding fragment thereof used for enrichment of the second CTNS signature peptide of cystinosis of SEQ ID NO: 7 or 8 comprises a VL domain of SEQ ID NO: 72.

31. The method of claim 21, wherein the antibody or antigen-binding fragment thereof used for enrichment of the second CTNS signature peptide of cystinosis of SEQ ID NO: 7 or 8 comprises a heavy chain of SEQ ID NO: 116.

32. The method of claim 21, wherein the antibody or antigen-binding fragment thereof used for enrichment of the second CTNS signature peptide of cystinosis of SEQ ID NO: 7 or 8 comprises a light chain of SEQ ID NO: 121.

33. The method of claim 21, wherein the antibody or antigen-binding fragment thereof used for enrichment of the first SHPK signature peptide of cystinosis of SEQ ID NO: 9 comprises a VH domain of SEQ ID NO: 73.

34. The method of claim 21, wherein the antibody or antigen-binding fragment thereof used for enrichment of the first SHPK signature peptide of cystinosis of SEQ ID NO: 9 comprises a VL domain of SEQ ID NO: 74.

35. The method of claim 21, wherein the antibody or antigen-binding fragment thereof used for enrichment of the first SHPK signature peptide of cystinosis of SEQ ID NO: 9 comprises a heavy chain of SEQ ID NO: 126.

36. The method of claim 21, wherein the antibody or antigen-binding fragment thereof used for enrichment of the first SHPK signature peptide of cystinosis of SEQ ID NO: 9 comprises a light chain of SEQ ID NO: 131.

37. The method of claim 1, wherein the method further comprises screening for Wilson Disease (WD) by enriching for:

a first ATP7B signature peptide of WD of SEQ ID NO: 10 with an antibody or antigen-binding fragment thereof that binds the first ATP7B signature peptide; and/or

a second ATP7B signature peptide of WD of SEQ ID NO: 11 or 21 with an antibody or antigen-binding fragment thereof that binds the second ATP7B signature peptide and comprises: a heavy chain variable (VH) domain comprising CDR1 of SEQ ID NO: 57, CDR2 of SEQ ID NO: 58, and CDR3 of SEQ ID NO: 59, and a light chain variable (VL) domain comprising: CDR1 of SEQ ID NO: 60, CDR2 of SEQ ID NO: 61, and CDR3 of SEQ ID NO: 62;

a third ATP7B signature peptide of WD of SEQ ID NO: 16 with an antibody or antigen-binding fragment thereof that binds the third ATP7B signature peptide;

a fourth ATP7B signature peptide of WD of SEQ ID NO: 17 with an antibody or antigen-binding fragment thereof that binds the fourth ATP7B signature peptide;

a fifth ATP7B signature peptide of WD of SEQ ID NO: 18 with an antibody or antigen-binding fragment thereof that binds the fifth ATP7B signature peptide;

a sixth ATP7B signature peptide of WD of SEQ ID NO: 19 with an antibody or antigen-binding fragment thereof that binds the sixth ATP7B signature peptide;

a seventh ATP7B signature peptide of WD of SEQ ID NO: 20 with an antibody or antigen-binding fragment thereof that binds the seventh ATP7B signature peptide; and

Diagnosing the subject with WD when the concentrations of the first ATP7B, the second ATP7B, the third ATP7B, the fourth ATP7B, the fifth ATP7B, the sixth ATP7B, and/or the seventh ATP7B signature peptides are lower than corresponding predetermined threshold concentrations or when the first ATP7B, the second ATP7B, the third ATP7B, the fourth ATP7B, the fifth ATP7B, the sixth ATP7B, and/or the seventh ATP7B signature peptides are absent.

38. The method of claim 37, wherein the corresponding predetermined threshold concentration for each signature peptide is calculated from a standard deviation of the mean concentration of each signature peptide in DBS from a population of normal control subjects.

39. The method of claim 38, wherein the mean concentration of the second ATP7B signature peptide of WD of SEQ ID NO: 11 or 21 in DBS from a population of normal control subjects comprises a concentration in a range of 90 pmol/L to 400 pmol/L.

40. The method of claim 38, wherein the mean concentration of the third ATP7B signature peptide of WD of SEQ ID NO: 16 in DBS from a population of normal control subjects comprises a concentration in a range of 30 pmol/L to 100 pmol/L.

41. The method of claim 38, wherein the mean concentration of the seventh ATP7B signature peptide of WD of SEQ ID NO: 20 in DBS from a population of normal control subjects comprises a concentration in a range of 200 pmol/L to 500 pmol/L.

42. The method of claim 37, wherein the antibody or antigen-binding fragment thereof used for enrichment of the second ATP7B signature peptide of WD of SEQ ID NO: 11 or 21 comprises a VH domain of SEQ ID NO: 75.

43. The method of claim 37, wherein the antibody or antigen-binding fragment thereof used for enrichment of the second ATP7B signature peptide of WD of SEQ ID NO: 11 or 21 comprises a VL domain of SEQ ID NO: 76.

44. The method of claim 37, wherein the antibody or antigen-binding fragment thereof used for enrichment of the second ATP7B signature peptide of WD of SEQ ID NO: 11 or 21 comprises a heavy chain of SEQ ID NO: 136.

45. The method of claim 37, wherein the antibody or antigen-binding fragment thereof used for enrichment of the second ATP7B signature peptide of WD of SEQ ID NO: 11 or 21 comprises a light chain of SEQ ID NO: 141.

46. A method of screening for severe combined immunodeficiency (SCID), Wiskott-Aldrich syndrome (WAS), and/or X-linked agammaglobulinemia (XLA) in a subject, the method comprising:

Obtaining a dried blood spot (DBS) sample derived from the subject;

Enriching for:

Enriching for an endogenous ATP7B peptide of:

SEQ ID NO: 10 with an antibody or antigen binding fragment thereof that binds the endogenous ATP7B peptide of SEQ ID NO: 10;

SEQ ID NO: 11 or 21 with an antibody or antigen-binding fragment thereof that binds the endogenous ATP7B peptide of SEQ ID NO: 11 or 21 and comprises: a heavy chain variable (VH) domain comprising CDR1 of SEQ ID NO: 57, CDR2 of SEQ ID NO: 58, and CDR3 of SEQ ID NO: 59, and a light chain variable (VL) domain comprising: CDR1 of SEQ ID NO: 60, CDR2 of SEQ ID NO: 61, and CDR3 of SEQ ID NO: 62;

SEQ ID NO: 16 with an antibody or antigen-binding fragment thereof that binds the endogenous ATP7B peptide of SEQ ID NO: 16;

SEQ ID NO: 17 with an antibody or antigen-binding fragment thereof that binds the endogenous ATP7B peptide of SEQ ID NO: 17;

SEQ ID NO: 18 with an antibody or antigen-binding fragment thereof that binds the endogenous ATP7B peptide of SEQ ID NO: 18;

SEQ ID NO: 19 with an antibody or antigen-binding fragment thereof that binds the endogenous ATP7B peptide of SEQ ID NO: 19;

SEQ ID NO: 20 with an antibody or antigen-binding fragment thereof that binds the endogenous ATP7B peptide of SEQ ID NO: 20; or

SEQ ID NO: 21 with an antibody or antigen-binding fragment thereof that binds the endogenous ATP7B peptide of SEQ ID NO: 21;

Performing liquid chromatography-multiple reaction monitoring mass spectrometry (LC-MRM-MS) on the enriched peptides to determine concentrations of the peptides;

Calculating a ratio of signature peptide concentration:endogenous ATP7B peptide concentration for each signature peptide; and

Diagnosing the subject with:

SCID when the ratio of CD3ε signature peptide concentration:endogenous ATP7B peptide concentration is lower than a corresponding predetermined threshold ratio or when the CD3ε signature peptide is absent;

WAS when the ratios of the first and/or second WASp signature peptide concentration:endogenous ATP7B peptide concentration are lower than corresponding predetermined threshold ratios or when the first and/or second WASp signature peptides are absent; and

XLA when the ratios of the first and/or second BTK signature peptide concentration:endogenous ATP7B peptide concentration are lower than corresponding predetermined threshold ratios or when the first and/or second BTK signature peptides are absent.

47. The method of claim 46, wherein each predetermined threshold ratio is calculated from standard deviation of the mean ratio of each peptide concentration:endogenous ATP7B peptide concentration in a population of samples.

48. The method of claim 46, wherein the method is performed as part of a newborn screening (NBS) that additionally screens the subject for one or more of phenylketonuria, primary congenital hypothyroidism, cystic fibrosis, and sickle cell disease.

49. The method of claim 46, wherein the method is performed in the absence of clinical symptoms of SCID, WAS, and/or XLA in the subject.

50. The method of claim 46, wherein the enzyme is trypsin.

51. The method of claim 46, wherein the antibody or antigen-binding fragment thereof used for enrichment of the CD3ε signature peptide of SCID of SEQ ID NO: 1 comprises a VH domain of SEQ ID NO: 63.

52. The method of claim 46, wherein the antibody or antigen-binding fragment thereof used for enrichment of the CD3ε signature peptide of SCID of SEQ ID NO: 1 comprises a VL domain of SEQ ID NO: 64.

53. The method of claim 46, wherein the antibody or antigen-binding fragment thereof used for enrichment of the first WASp signature peptide of WAS of SEQ ID NO: 2 comprises a VH domain of SEQ ID NO: 65.

54. The method of claim 46, wherein the antibody or antigen-binding fragment thereof used for enrichment of the first WASp signature peptide of WAS of SEQ ID NO: 2 comprises a VL domain of SEQ ID NO: 66.

55. The method of claim 46, wherein the antibody or antigen-binding fragment thereof used for enrichment of the first WASp signature peptide of WAS of SEQ ID NO: 2 comprises a heavy chain of SEQ ID NO: 86.

56. The method of claim 46, wherein the antibody or antigen-binding fragment thereof used for enrichment of the first WASp signature peptide of WAS of SEQ ID NO: 2 comprises a light chain of SEQ ID NO: 91.

57. The method of claim 46, wherein the antibody or antigen-binding fragment thereof used for enrichment of the first BTK signature peptide of XLA of SEQ ID NO: 4 comprises a VH domain of SEQ ID NO: 67.

58. The method of claim 46, wherein the antibody or antigen-binding fragment thereof used for enrichment of the first BTK signature peptide of XLA of SEQ ID NO: 4 comprises a VL domain of SEQ ID NO: 68.

59. The method of claim 46, wherein the antibody or antigen-binding fragment thereof used for enrichment of the first BTK signature peptide of XLA of SEQ ID NO: 4 comprises a heavy chain of SEQ ID NO: 96.

60. The method of claim 46, wherein the antibody or antigen-binding fragment thereof used for enrichment of the first BTK signature peptide of XLA of SEQ ID NO: 4 comprises a light chain of SEQ ID NO: 101.

61. The method of claim 46, further comprising screening for cystinosis by enriching for:

a second SHPK signature peptide of cystinosis of SEQ ID NO: 14 with an antibody or antigen-binding fragment thereof that binds the second SHPK signature peptide; and/or

Diagnosing the subject with cystinosis when the ratio of the first CTNS peptide concentration:endogenous ATP7B peptide concentration, the second CTNS peptide concentration:endogenous ATP7B peptide concentration, the third CTNS peptide concentration:endogenous ATP7B peptide concentration, the fourth CTNS peptide concentration:endogenous ATP7B peptide concentration, the first SHPK peptide concentration:endogenous ATP7B peptide concentration, the second SHPK peptide concentration:endogenous ATP7B peptide concentration, and/or the third SHPK signature peptide concentration:endogenous ATP7B peptide concentration is lower than a corresponding predetermined threshold ratio or when the first CTNS, the second CTNS, the third CTNS, the fourth CTNS, the first SHPK, the second SHPK, and/or the third SHPK signature peptides are absent.

62. The method of claim 61, wherein the antibody or antigen-binding fragment thereof used for enrichment of the first CTNS signature peptide of cystinosis of SEQ ID NO: 6 comprises a VH domain of SEQ ID NO: 69.

63. The method of claim 61, wherein the antibody or antigen-binding fragment thereof used for enrichment of the first CTNS signature peptide of cystinosis of SEQ ID NO: 6 comprises a VL domain of SEQ ID NO: 70.

64. The method of claim 61, wherein the antibody or antigen-binding fragment thereof used for enrichment of the first CTNS signature peptide of cystinosis of SEQ ID NO: 6 comprises a heavy chain of SEQ ID NO: 106.

65. The method of claim 61, wherein the antibody or antigen-binding fragment thereof used for enrichment of the first CTNS signature peptide of cystinosis of SEQ ID NO: 6 comprises a light chain of SEQ ID NO: 111.

66. The method of claim 61, wherein the antibody or antigen-binding fragment thereof used for enrichment of the second CTNS signature peptide of cystinosis of SEQ ID NO: 7 or 8 comprises a VH domain of SEQ ID NO: 71.

67. The method of claim 61, wherein the antibody or antigen-binding fragment thereof used for enrichment of the second CTNS signature peptide of cystinosis of SEQ ID NO: 7 or 8 comprises a VL domain of SEQ ID NO: 72.

68. The method of claim 61, wherein the antibody or antigen-binding fragment thereof used for enrichment of the second CTNS signature peptide of cystinosis of SEQ ID NO: 7 or 8 comprises a heavy chain of SEQ ID NO: 116.

69. The method of claim 61, wherein the antibody or antigen-binding fragment thereof used for enrichment of the second CTNS signature peptide of cystinosis of SEQ ID NO: 7 or 8 comprises a light chain of SEQ ID NO: 121.

70. The method of claim 61, wherein the antibody or antigen-binding fragment thereof used for enrichment of the first SHPK signature peptide of cystinosis of SEQ ID NO: 9 comprises a VH domain of SEQ ID NO: 73.

71. The method of claim 61, wherein the antibody or antigen-binding fragment thereof used for enrichment of the first SHPK signature peptide of cystinosis of SEQ ID NO: 9 comprises a VL domain of SEQ ID NO: 74.

72. The method of claim 61, wherein the antibody or antigen-binding fragment thereof used for enrichment of the first SHPK signature peptide of cystinosis of SEQ ID NO: 9 comprises a heavy chain of SEQ ID NO: 126.

73. The method of claim 61, wherein the antibody or antigen-binding fragment thereof used for enrichment of the first SHPK signature peptide of cystinosis of SEQ ID NO: 9 comprises a light chain of SEQ ID NO: 131.

74. The method of claim 61, wherein the antibody or antigen-binding fragment thereof used for enrichment of the endogenous ATP7B peptide of SEQ ID NO: 11 or 21 comprises a VH domain of SEQ ID NO: 75.

75. The method of claim 61, wherein the antibody or antigen-binding fragment thereof used for enrichment of the endogenous ATP7B signature peptide of SEQ ID NO: 11 or 21 comprises a VL domain of SEQ ID NO: 76.

76. The method of claim 61, wherein the antibody or antigen-binding fragment thereof used for enrichment of the endogenous ATP7B signature peptide of SEQ ID NO: 11 or 21 comprises a heavy chain of SEQ ID NO: 136.

77. The method of claim 61, wherein the antibody or antigen-binding fragment thereof used for enrichment of the endogenous ATP7B signature peptide of SEQ ID NO: 11 or 21 comprises a light chain of SEQ ID NO: 141.

78. A method of detecting one or more signature peptides of severe combined immunodeficiency (SCID), Wiskott-Aldrich syndrome (WAS), and/or X-linked agammaglobulinemia (XLA) in one or more dried blood spot (DBS) samples, the method comprising:

Obtaining the one or more dried blood spot (DBS) samples;

Digesting proteins from blood of each DBS with an enzyme to yield one or more peptides;

Enriching for:

Performing liquid chromatography-multiple reaction monitoring mass spectrometry (LC-MRM-MS) on the enriched peptides, thus detecting one or more signature peptides of SCID, WAS, and XLA in one or more DBS samples.

79. The method of claim 78, wherein the enzyme is trypsin.

80. The method of claim 78, wherein the antibody or antigen-binding fragment thereof used for enrichment of the CD3ε signature peptide of SCID of SEQ ID NO: 1 comprises a VH domain of SEQ ID NO: 63.

81. The method of claim 78, wherein the antibody or antigen-binding fragment thereof used for enrichment of the CD3ε signature peptide of SCID of SEQ ID NO: 1 comprises a VL domain of SEQ ID NO: 64.

82. The method of claim 78, wherein the antibody or antigen-binding fragment thereof used for enrichment of the first WASp signature peptide of WAS of SEQ ID NO: 2 comprises a VH domain of SEQ ID NO: 65.

83. The method of claim 78, wherein the antibody or antigen-binding fragment thereof used for enrichment of the first WASp signature peptide of WAS of SEQ ID NO: 2 comprises a VL domain of SEQ ID NO: 66.

84. The method of claim 78, wherein the antibody or antigen-binding fragment thereof used for enrichment of the first WASp signature peptide of WAS of SEQ ID NO: 2 comprises a heavy chain of SEQ ID NO: 86.

85. The method of claim 78, wherein the antibody or antigen-binding fragment thereof used for enrichment of the first WASp signature peptide of WAS of SEQ ID NO: 2 comprises a light chain of SEQ ID NO: 91.

86. The method of claim 78, wherein the antibody or antigen-binding fragment thereof used for enrichment of the first BTK signature peptide of XLA of SEQ ID NO: 4 comprises a VH domain of SEQ ID NO: 67.

87. The method of claim 78, wherein the antibody or antigen-binding fragment thereof used for enrichment of the first BTK signature peptide of XLA of SEQ ID NO: 4 comprises a VL domain of SEQ ID NO: 68.

88. The method of claim 78, wherein the antibody or antigen-binding fragment thereof used for enrichment of the first BTK signature peptide of XLA of SEQ ID NO: 4 comprises a heavy chain of SEQ ID NO: 96.

89. The method of claim 78, wherein the antibody or antigen-binding fragment thereof used for enrichment of the first BTK signature peptide of XLA of SEQ ID NO: 4 comprises a light chain of SEQ ID NO: 101.

90. The method of claim 78, further comprising detecting one or more signature peptides of cystinosis by enriching for:

Performing liquid chromatography-multiple reaction monitoring mass spectrometry (LC-MRM-MS) on the enriched peptides, thus detecting one or more signature peptides of cystinosis in one or more DBS samples.

91. The method of claim 90, wherein the antibody or antigen-binding fragment thereof used for enrichment of the first CTNS signature peptide of cystinosis of SEQ ID NO: 6 comprises a VH domain of SEQ ID NO: 69.

92. The method of claim 90, wherein the antibody or antigen-binding fragment thereof used for enrichment of the first CTNS signature peptide of cystinosis of SEQ ID NO: 6 comprises a VL domain of SEQ ID NO: 70.

93. The method of claim 90, wherein the antibody or antigen-binding fragment thereof used for enrichment of the first CTNS signature peptide of cystinosis of SEQ ID NO: 6 comprises a heavy chain of SEQ ID NO: 106.

94. The method of claim 90, wherein the antibody or antigen-binding fragment thereof used for enrichment of the first CTNS signature peptide of cystinosis of SEQ ID NO: 6 comprises a light chain of SEQ ID NO: 111.

95. The method of claim 90, wherein the antibody or antigen-binding fragment thereof used for enrichment of the second CTNS signature peptide of cystinosis of SEQ ID NO: 7 or 8 comprises a VH domain of SEQ ID NO: 71.

96. The method of claim 90, wherein the antibody or antigen-binding fragment thereof used for enrichment of the second CTNS signature peptide of cystinosis of SEQ ID NO: 7 or 8 comprises a VL domain of SEQ ID NO: 72.

97. The method of claim 90, wherein the antibody or antigen-binding fragment thereof used for enrichment of the second CTNS signature peptide of cystinosis of SEQ ID NO: 7 or 8 comprises a heavy chain of SEQ ID NO: 116.

98. The method of claim 90, wherein the antibody or antigen-binding fragment thereof used for enrichment of the second CTNS signature peptide of cystinosis of SEQ ID NO: 7 or 8 comprises a light chain of SEQ ID NO: 121.

99. The method of claim 90, wherein the antibody or antigen-binding fragment thereof used for enrichment of the first SHPK signature peptide of cystinosis of SEQ ID NO: 9 comprises a VH domain of SEQ ID NO: 73.

100. The method of claim 90, wherein the antibody or antigen-binding fragment thereof used for enrichment of the first SHPK signature peptide of cystinosis of SEQ ID NO: 9 comprises a VL domain of SEQ ID NO: 74.

101. The method of claim 90, wherein the antibody or antigen-binding fragment thereof used for enrichment of the first SHPK signature peptide of cystinosis of SEQ ID NO: 9 comprises a heavy chain of SEQ ID NO: 126.

102. The method of claim 90, wherein the antibody or antigen-binding fragment thereof used for enrichment of the first SHPK signature peptide of cystinosis of SEQ ID NO: 9 comprises a light chain of SEQ ID NO: 131.

103. The method of claim 78, further comprising detecting one or more signature peptides of Wilson Disease (WD) by enriching for:

a sixth ATP7B signature peptide of WD of SEQ ID NO: 19 with an antibody or antigen-binding fragment thereof that binds the sixth ATP7B signature peptide; and/or

a seventh ATP7B signature peptide of WD of SEQ ID NO: 20 with an antibody or antigen-binding fragment thereof that binds the seventh ATP7B signature peptide;

Performing liquid chromatography-multiple reaction monitoring mass spectrometry (LC-MRM-MS) on the enriched peptides, thus detecting one or more signature peptides of WD in one or more DBS samples.

104. The method of claim 103, wherein the antibody or antigen-binding fragment thereof used for enrichment of the second ATP7B signature peptide of WD of SEQ ID NO: 11 or 21 comprises a VH domain of SEQ ID NO: 75.

105. The method of claim 103, wherein the antibody or antigen-binding fragment thereof used for enrichment of the second ATP7B signature peptide of WD of SEQ ID NO: 11 or 21 comprises a VL domain of SEQ ID NO: 76.

106. The method of claim 103, wherein the antibody or antigen-binding fragment thereof used for enrichment of the second ATP7B signature peptide of WD of SEQ ID NO: 11 or 21 comprises a heavy chain of SEQ ID NO: 136.

107. The method of claim 103, wherein the antibody or antigen-binding fragment thereof used for enrichment of the second ATP7B signature peptide of WD of SEQ ID NO: 11 or 21 comprises a light chain of SEQ ID NO: 141.

108. An assay for the screening of severe combined immunodeficiency (SCID), Wiskott-Aldrich syndrome (WAS), X-linked agammaglobulinemia (XLA) in a subject, and/or Wilson Disease (WD), the assay comprising:

(i) an antibody or antigen-binding fragment thereof comprising:

a heavy chain variable (VH) domain comprising CDR1 of SEQ ID NO: 22, CDR2 of SEQ ID NO: 23, and CDR3 of SEQ ID NO: 24, and a light chain variable (VL) domain comprising: CDR1 of SEQ ID NO: 25, CDR2 of SEQ ID NO: 26, and CDR3 of SEQ ID NO: 27 that binds to a CD3ε signature peptide of SCID of SEQ ID NO: 1;

a heavy chain variable (VH) domain comprising CDR1 of SEQ ID NO: 28, CDR2 of SEQ ID NO: 29, and CDR3 of SEQ ID NO: 30, and a light chain variable (VL) domain comprising: CDR1 of SEQ ID NO: 31, CDR2 of SEQ ID NO: 32, and CDR3 of SEQ ID NO: 33 that binds to a WASp signature peptide of WAS of SEQ ID NO: 2;

a heavy chain variable (VH) domain comprising CDR1 of SEQ ID NO: 34, CDR2 of SEQ ID NO: 35, and CDR3 of sequence GDI, and a light chain variable (VL) domain comprising: CDR1 of SEQ ID NO: 36, CDR2 of SEQ ID NO: 37, and CDR3 of SEQ ID NO: 38 that binds to a BTK signature peptide of XLA of SEQ ID NO: 4; and/or

a heavy chain variable (VH) domain comprising CDR1 of SEQ ID NO: 57, CDR2 of SEQ ID NO: 58, and CDR3 of SEQ ID NO: 59, and a light chain variable (VL) domain comprising: CDR1 of SEQ ID NO: 60, CDR2 of SEQ ID NO: 61, and CDR3 of SEQ ID NO: 62 that binds to an ATP7B peptide of SEQ ID NO: 11 or 21;

and/or

(ii) an antibody or antigen-binding fragment thereof that binds a WASp signature peptide of WAS of SEQ ID NO: 3;

an antibody or antigen-binding fragment thereof that binds a BTK signature peptide of XLA of SEQ ID NO: 5;

an antibody or antigen-binding fragment thereof that binds an ATP7B signature peptide of SEQ ID NO: 10;

an antibody or antigen-binding fragment thereof that binds an ATP7B signature peptide of SEQ ID NO: 16;

an antibody or antigen-binding fragment thereof that binds an ATP7B signature peptide of SEQ ID NO: 17;

an antibody or antigen-binding fragment thereof that binds an ATP7B signature peptide of SEQ ID NO: 18;

an antibody or antigen-binding fragment thereof that binds an ATP7B signature peptide of SEQ ID NO: 19;

an antibody or antigen-binding fragment thereof that binds an ATP7B signature peptide of SEQ ID NO: 20; and/or

an antibody or antigen-binding fragment thereof that binds an ATP7B signature peptide of SEQ ID NO: 21;

and/or

(iii) reference signature peptides comprising:

a CD3ε peptide of SCID of SEQ ID NO: 1;

a WASp peptide of WAS of SEQ ID NO: 2;

a WASp peptide of WAS of SEQ ID NO: 3;

a BTK peptide of XLA of SEQ ID NO: 4;

a BTK peptide of XLA of SEQ ID NO: 5;

an ATP7B peptide of WD of SEQ ID NO: 10;

an ATP7B peptide of WD of SEQ ID NO: 11;

an ATP7B peptide of WD of SEQ ID NO: 16;

an ATP7B peptide of WD of SEQ ID NO: 17;

an ATP7B peptide of WD of SEQ ID NO: 18;

an ATP7B peptide of WD of SEQ ID NO: 19;

an ATP7B peptide of WD of SEQ ID NO: 20; and/or

an ATP7B peptide of WD of SEQ ID NO: 21.

109. The assay of claim 108, wherein the antibody or antigen-binding fragment thereof that binds to the CD3ε signature peptide of SCID of SEQ ID NO: 1 comprises a VH domain of SEQ ID NO: 63.

110. The assay of claim 108, wherein the antibody or antigen-binding fragment thereof that binds to the CD3ε signature peptide of SCID of SEQ ID NO: 1 comprises a VL domain of SEQ ID NO: 64.

111. The assay of claim 108, wherein the antibody or antigen-binding fragment thereof that binds to the WASp signature peptide of WAS of SEQ ID NO: 2 comprises a VH domain of SEQ ID NO: 65.

112. The assay of claim 108, wherein the antibody or antigen-binding fragment thereof that binds to the WASp signature peptide of WAS of SEQ ID NO: 2 comprises a VL domain of SEQ ID NO: 66.

113. The assay of claim 108, wherein the antibody or antigen-binding fragment thereof that binds to the WASp signature peptide of WAS of SEQ ID NO: 2 comprises a heavy chain of SEQ ID NO: 86.

114. The assay of claim 108, wherein the antibody or antigen-binding fragment thereof that binds to the WASp signature peptide of WAS of SEQ ID NO: 2 comprises a light chain of SEQ ID NO: 91.

115. The assay of claim 108, wherein the antibody or antigen-binding fragment thereof that binds to the BTK signature peptide of XLA of SEQ ID NO: 4 comprises a VH domain of SEQ ID NO: 67.

116. The assay of claim 108, wherein the antibody or antigen-binding fragment thereof that binds to the BTK signature peptide of XLA of SEQ ID NO: 4 comprises a VL domain of SEQ ID NO: 68.

117. The assay of claim 108, wherein the antibody or antigen-binding fragment thereof that binds to the BTK signature peptide of XLA of SEQ ID NO: 4 comprises a heavy chain of SEQ ID NO: 96.

118. The assay of claim 108, wherein the antibody or antigen-binding fragment thereof that binds to the BTK signature peptide of XLA of SEQ ID NO: 4 comprises a light chain of SEQ ID NO: 101.

119. The assay of claim 108 to further screen for cystinosis, comprising:

(iii) an antibody or antigen-binding fragment thereof comprising:

a heavy chain variable (VH) domain comprising CDR1 of SEQ ID NO: 39, CDR2 of SEQ ID NO: 40, and CDR3 of SEQ ID NO: 41, and a light chain variable (VL) domain comprising: CDR1 of SEQ ID NO: 42, CDR2 of SEQ ID NO: 43, and CDR3 of SEQ ID NO: 44 that binds to a CTNS signature peptide of cystinosis of SEQ ID NO: 6;

a heavy chain variable (VH) domain comprising CDR1 of SEQ ID NO: 45, CDR2 of SEQ ID NO: 46, and CDR3 of SEQ ID NO: 47, and a light chain variable (VL) domain comprising: CDR1 of SEQ ID NO: 48, CDR2 of SEQ ID NO: 49, and CDR3 of SEQ ID NO: 50 that binds to a CTNS signature peptide of cystinosis of SEQ ID NO: 7 or 8;

a heavy chain variable (VH) domain comprising CDR1 of SEQ ID NO: 51, CDR2 of SEQ ID NO: 52, and CDR3 of SEQ ID NO: 53, and a light chain variable (VL) domain comprising: CDR1 of SEQ ID NO: 54, CDR2 of SEQ ID NO: 55, and CDR3 of SEQ ID NO: 56 that binds to a SHPK signature peptide of cystinosis of SEQ ID NO: 9;

and/or

(iv) an antibody or antigen-binding fragment thereof that binds a CTNS signature peptide of SEQ ID NO: 12;

an antibody or antigen-binding fragment thereof that binds a CTNS signature peptide of SEQ ID NO: 13;

an antibody or antigen-binding fragment thereof that binds a SHPK signature peptide of SEQ ID NO: 14; and/or

an antibody or antigen-binding fragment thereof that binds a SHPK signature peptide of SEQ ID NO: 15;

and/or

(v) reference signature peptides comprising:

a CTNS peptide of cystinosis of SEQ ID NO: 6;

a CTNS peptide of cystinosis of SEQ ID NO: 7 or 8;

a CTNS peptide of cystinosis of SEQ ID NO: 12;

a CTNS peptide of cystinosis of SEQ ID NO: 13;

a SHPK peptide of cystinosis of SEQ ID NO: 9;

a SHPK peptide of cystinosis of SEQ ID NO: 14; and/or

a SHPK peptide of cystinosis of SEQ ID NO: 15.

120. The assay of claim 119, wherein the antibody or antigen-binding fragment thereof that binds to the CTNS signature peptide of cystinosis of SEQ ID NO: 6 comprises a VH domain of SEQ ID NO: 69.

121. The assay of claim 119, wherein the antibody or antigen-binding fragment thereof that binds to the CTNS signature peptide of cystinosis of SEQ ID NO: 6 comprises a VL domain of SEQ ID NO: 70.

122. The assay of claim 119, wherein the antibody or antigen-binding fragment thereof that binds to the CTNS signature peptide of cystinosis of SEQ ID NO: 6 comprises a heavy chain of SEQ ID NO: 106.

123. The assay of claim 119, wherein the antibody or antigen-binding fragment thereof that binds to the CTNS signature peptide of cystinosis of SEQ ID NO: 6 comprises a light chain of SEQ ID NO: 111.

124. The assay of claim 119, wherein the antibody or antigen-binding fragment thereof that binds to the CTNS signature peptide of cystinosis of SEQ ID NO: 7 or 8 comprises a VH domain of SEQ ID NO: 71.

125. The assay of claim 119, wherein the antibody or antigen-binding fragment thereof that binds to the CTNS signature peptide of cystinosis of SEQ ID NO: 7 or 8 comprises a VL domain of SEQ ID NO: 72.

126. The assay of claim 119, wherein the antibody or antigen-binding fragment thereof that binds to the CTNS signature peptide of cystinosis of SEQ ID NO: 7 or 8 comprises a heavy chain of SEQ ID NO: 116.

127. The assay of claim 119, wherein the antibody or antigen-binding fragment thereof that binds to the CTNS signature peptide of cystinosis of SEQ ID NO: 7 or 8 comprises a light chain of SEQ ID NO: 121.

128. The assay of claim 119, wherein the antibody or antigen-binding fragment thereof that binds to the SHPK signature peptide of cystinosis of SEQ ID NO: 9 comprises a VH domain of SEQ ID NO: 73.

129. The assay of claim 119, wherein the antibody or antigen-binding fragment thereof that binds to the SHPK signature peptide of cystinosis of SEQ ID NO: 9 comprises a VL domain of SEQ ID NO: 74.

130. The assay of claim 119, wherein the antibody or antigen-binding fragment thereof that binds to the SHPK signature peptide of cystinosis of SEQ ID NO: 9 comprises a heavy chain of SEQ ID NO: 126.

131. The assay of claim 119, wherein the antibody or antigen-binding fragment thereof that binds to the SHPK signature peptide of cystinosis of SEQ ID NO: 9 comprises a light chain of SEQ ID NO: 131.

132. The assay of claim 119, wherein the antibody or antigen-binding fragment thereof that binds to the ATP7B signature peptide of WD of SEQ ID NO: 11 or 21 comprises a VH domain of SEQ ID NO: 75.

133. The assay of claim 119, wherein the antibody or antigen-binding fragment thereof that binds to the ATP7B signature peptide of WD of SEQ ID NO: 11 or 21 comprises a VL domain of SEQ ID NO: 76.

134. The assay of claim 119, wherein the antibody or antigen-binding fragment thereof that binds to the ATP7B signature peptide of WD of SEQ ID NO: 11 or 21 comprises a heavy chain of SEQ ID NO: 136.

135. The assay of claim 119, wherein the antibody or antigen-binding fragment thereof that binds to the ATP7B signature peptide of WD of SEQ ID NO: 11 or 21 comprises a light chain of SEQ ID NO: 141.

136. The assay of claim 119, wherein the reference signature peptides are isotopically labeled.

137. The assay of claim 119, wherein the antibodies or antigen-binding fragments thereof are attached to magnetic beads.

138. An isolated antibody or antigen binding fragment thereof comprising: a heavy chain variable (VH) domain comprising CDR1 of SEQ ID NO: 22, CDR2 of SEQ ID NO: 23, and CDR3 of SEQ ID NO: 24, and a light chain variable (VL) domain comprising: CDR1 of SEQ ID NO: 25, CDR2 of SEQ ID NO: 26, and CDR3 of SEQ ID NO: 27.

139. The isolated antibody or antigen binding fragment thereof of claim 138, wherein the VH domain comprises SEQ ID NO: 63.

140. The isolated antibody or antigen binding fragment thereof of claim 138, wherein the VL domain comprises SEQ ID NO: 64.

141. An isolated antibody or antigen binding fragment thereof comprising: a heavy chain variable (VH) domain comprising CDR1 of SEQ ID NO: 28, CDR2 of SEQ ID NO: 29, and CDR3 of SEQ ID NO: 30, and a light chain variable (VL) domain comprising: CDR1 of SEQ ID NO: 31, CDR2 of SEQ ID NO: 32, and CDR3 of SEQ ID NO: 33.

142. The isolated antibody or antigen binding fragment thereof of claim 141, wherein the VH domain comprises SEQ ID NO: 65.

143. The isolated antibody or antigen binding fragment thereof of claim 141, wherein the VL domain comprises SEQ ID NO: 66.

144. The isolated antibody or antigen binding fragment thereof of claim 141, wherein the heavy chain comprises SEQ ID NO: 86.

145. The isolated antibody or antigen binding fragment thereof of claim 141, wherein the light chain comprises SEQ ID NO: 91.

146. An isolated antibody or antigen binding fragment thereof comprising: a heavy chain variable (VH) domain comprising CDR1 of SEQ ID NO: 34, CDR2 of SEQ ID NO: 35, and CDR3 of sequence GDI, and a light chain variable (VL) domain comprising: CDR1 of SEQ ID NO: 36, CDR2 of SEQ ID NO: 37, and CDR3 of SEQ ID NO: 38.

147. The isolated antibody or antigen binding fragment thereof of claim 146, wherein the VH domain comprises SEQ ID NO: 67.

148. The isolated antibody or antigen binding fragment thereof of claim 146, wherein the VL domain comprises SEQ ID NO: 68.

149. The isolated antibody or antigen binding fragment thereof of claim 146, wherein the heavy chain comprises SEQ ID NO: 96.

150. The isolated antibody or antigen binding fragment thereof of claim 146, wherein the light chain comprises SEQ ID NO: 101.

151. An isolated antibody or antigen binding fragment thereof comprising: a heavy chain variable (VH) domain comprising CDR1 of SEQ ID NO: 39, CDR2 of SEQ ID NO: 40, and CDR3 of SEQ ID NO: 41, and a light chain variable (VL) domain comprising: CDR1 of SEQ ID NO: 42, CDR2 of SEQ ID NO: 43, and CDR3 of SEQ ID NO: 44.

152. The isolated antibody or antigen binding fragment thereof of claim 151, wherein the VH domain comprises SEQ ID NO: 69.

153. The isolated antibody or antigen binding fragment thereof of claim 151, wherein the VL domain comprises SEQ ID NO: 70.

154. The isolated antibody or antigen binding fragment thereof of claim 151, wherein the heavy chain comprises SEQ ID NO: 106.

155. The isolated antibody or antigen binding fragment thereof of claim 151, wherein the light chain comprises SEQ ID NO: 111.

156. An isolated antibody or antigen binding fragment thereof comprising: a heavy chain variable (VH) domain comprising CDR1 of SEQ ID NO: 45, CDR2 of SEQ ID NO: 46, and CDR3 of SEQ ID NO: 47, and a light chain variable (VL) domain comprising: CDR1 of SEQ ID NO: 48, CDR2 of SEQ ID NO: 49, and CDR3 of SEQ ID NO: 50.

157. The isolated antibody or antigen binding fragment thereof of claim 156, wherein the VH domain comprises SEQ ID NO: 71.

158. The isolated antibody or antigen binding fragment thereof of claim 156, wherein the VL domain comprises SEQ ID NO: 72.

159. The isolated antibody or antigen binding fragment thereof of claim 156, wherein the heavy chain comprises SEQ ID NO: 116.

160. The isolated antibody or antigen binding fragment thereof of claim 156, wherein the light chain comprises SEQ ID NO: 121.

161. An isolated antibody or antigen binding fragment thereof comprising: a heavy chain variable (VH) domain comprising CDR1 of SEQ ID NO: 51, CDR2 of SEQ ID NO: 52, and CDR3 of SEQ ID NO: 53, and a light chain variable (VL) domain comprising: CDR1 of SEQ ID NO: 54, CDR2 of SEQ ID NO: 55, and CDR3 of SEQ ID NO: 56.

162. The isolated antibody or antigen binding fragment thereof of claim 161, wherein the VH domain comprises SEQ ID NO: 73.

163. The isolated antibody or antigen binding fragment thereof of claim 161, wherein the VL domain comprises SEQ ID NO: 74.

164. The isolated antibody or antigen binding fragment thereof of claim 161, wherein the heavy chain comprises SEQ ID NO: 126.

165. The isolated antibody or antigen binding fragment thereof of claim 161, wherein the light chain comprises SEQ ID NO: 131.

166. An isolated antibody or antigen binding fragment thereof comprising: a heavy chain variable (VH) domain comprising CDR1 of SEQ ID NO: 57, CDR2 of SEQ ID NO: 58, and CDR3 of SEQ ID NO: 59, and a light chain variable (VL) domain comprising: CDR1 of SEQ ID NO: 60, CDR2 of SEQ ID NO: 61, and CDR3 of SEQ ID NO: 62.

167. The isolated antibody or antigen binding fragment thereof of claim 166, wherein the VH domain comprises SEQ ID NO: 75.

168. The isolated antibody or antigen binding fragment thereof of claim 166, wherein the VL domain comprises SEQ ID NO: 76.

169. The isolated antibody or antigen binding fragment thereof of claim 166, wherein the heavy chain comprises SEQ ID NO: 136.

170. The isolated antibody or antigen binding fragment thereof of claim 166, wherein the light chain comprises SEQ ID NO: 141.

171. A kit comprising:

(i) an antibody or antigen-binding fragment thereof comprising:

a heavy chain variable (VH) domain comprising CDR1 of SEQ ID NO: 34, CDR2 of SEQ ID NO: 35, and CDR3 of sequence GDI, and a light chain variable (VL) domain comprising: CDR1 of SEQ ID NO: 36, CDR2 of SEQ ID NO: 37, and CDR3 of SEQ ID NO: 38 that binds to a BTK signature peptide of XLA of SEQ ID NO: 4;

a heavy chain variable (VH) domain comprising CDR1 of SEQ ID NO: 51, CDR2 of SEQ ID NO: 52, and CDR3 of SEQ ID NO: 53, and a light chain variable (VL) domain comprising: CDR1 of SEQ ID NO: 54, CDR2 of SEQ ID NO: 55, and CDR3 of SEQ ID NO: 56 that binds to a SHPK signature peptide of cystinosis of SEQ ID NO: 9; and/or

and/or

an antibody or antigen-binding fragment thereof that binds a CTNS signature peptide of SEQ ID NO: 12;

an antibody or antigen-binding fragment thereof that binds a SHPK signature peptide of SEQ ID NO: 14;

and/or

(iii) reference signature peptides comprising:

a CD3ε peptide of SCID of SEQ ID NO: 1;

a WASp peptide of WAS of SEQ ID NO: 2;

a WASp peptide of WAS of SEQ ID NO: 3;

a BTK peptide of XLA of SEQ ID NO: 4;

a BTK peptide of XLA of SEQ ID NO: 5;

a CTNS peptide of cystinosis of SEQ ID NO: 6;

a CTNS peptide of cystinosis of SEQ ID NO: 7 or 8;

a CTNS peptide of cystinosis of SEQ ID NO: 12;

a CTNS peptide of cystinosis of SEQ ID NO: 13;

a SHPK peptide of cystinosis of SEQ ID NO: 9;

a SHPK peptide of cystinosis of SEQ ID NO: 14;

a SHPK peptide of cystinosis of SEQ ID NO: 15;

an ATP7B peptide of WD of SEQ ID NO: 10;

an ATP7B peptide of WD of SEQ ID NO: 11;

an ATP7B peptide of WD of SEQ ID NO: 16;

an ATP7B peptide of WD of SEQ ID NO: 17;

an ATP7B peptide of WD of SEQ ID NO: 18;

an ATP7B peptide of WD of SEQ ID NO: 19;

an ATP7B peptide of WD of SEQ ID NO: 20; and/or

an ATP7B peptide of WD of SEQ ID NO: 21.

172. The kit of claim 171, further comprising one or more of filter paper card, punch tool, digestion enzymes, digestion buffers, solid support for the antibodies or antigen-binding fragments thereof; and elution buffers.

173. The kit of claim 171, wherein the reference signature peptides are isotopically labeled.

174. The kit of claim 171, wherein the antibodies or antigen-binding fragments thereof are attached to magnetic beads.

175. A method of screening for severe combined immunodeficiency (SCID), Wiskott-Aldrich syndrome (WAS), X-linked agammaglobulinemia (XLA), cystinosis, and/or Wilson disease (WD) in a subject, the method comprising:

Obtaining a dried blood spot (DBS) sample derived from the subject;

Enriching for:

a CD3ε signature peptide of SCID with an antibody or antigen-binding fragment thereof that binds to the CD3ε signature peptide;

a first WASp signature peptide of WAS with an antibody or antigen-binding fragment thereof that binds to the first WASp signature peptide;

a second WASp signature peptide of WAS with an antibody or antigen-binding fragment thereof that binds to the second WASp signature peptide;

a first BTK signature peptide of XLA with an antibody or antigen-binding fragment thereof that binds to the first BTK signature peptide;

a second BTK signature peptide with an antibody or antigen-binding fragment thereof that binds to the second BTK signature peptide;

a first CTNS signature peptide of cystinosis with an antibody or antigen-binding fragment thereof that binds to the first CTNS signature peptide;

a second CTNS signature peptide of cystinosis with an antibody or antigen-binding fragment thereof that binds to the second CTNS signature peptide;

a third CTNS signature peptide of cystinosis with an antibody or antigen-binding fragment thereof that binds to the third CTNS signature peptide;

a fourth CTNS signature peptide of cystinosis with an antibody or antigen-binding fragment thereof that binds to the fourth CTNS signature peptide;

a first SHPK signature peptide of cystinosis with an antibody or antigen-binding fragment thereof that binds to the first SHPK signature peptide;

a second SHPK signature peptide of cystinosis with an antibody or antigen-binding fragment thereof that binds to the second SHPK signature peptide; and/or

a third SHPK signature peptide of cystinosis with an antibody or antigen-binding fragment thereof that binds to the third SHPK signature peptide;

a first ATP7B signature peptide with an antibody or antigen-binding fragment thereof that binds to the first ATP7B signature peptide;

a second ATP7B signature peptide with an antibody or antigen-binding fragment thereof that binds to the second ATP7B signature peptide;

a third ATP7B signature peptide with an antibody or antigen-binding fragment that binds to the third ATP7B signature peptide;

a fourth ATP7B signature peptide with an antibody or antigen-binding fragment thereof that binds to the fourth ATP7B signature peptide;

a fifth ATP7B signature peptide with an antibody or antigen-binding fragment thereof that binds to the fifth ATP7B signature peptide;

a sixth ATP7B signature peptide with an antibody or antigen-binding fragment thereof that binds to the sixth ATP7B signature peptide; and/or

a seventh ATP7B signature peptide with an antibody or antigen-binding fragment thereof;

Diagnosing the subject with:

WAS when the concentrations of the first and/or second WASp signature peptides are lower than corresponding predetermined threshold concentrations or when the first and/or second WASp signature peptides are absent;

XLA when the concentrations of the first and/or second BTK signature peptides are lower than corresponding predetermined threshold concentrations or when the first and/or second BTK signature peptides are absent;

cystinosis when the concentrations of the first CTNS, the second CTNS, the third CTNS, the fourth CTNS, the first SHPK, the second SHPK, and/or the third SHPK signature peptides are lower than corresponding predetermined threshold concentrations or when the first CTNS, the second CTNS, the third CTNS, the fourth CTNS, the first SHPK, the second SHPK, and/or the third SHPK signature peptides are absent; and/or

WD when the concentrations of the first ATP7B, the second ATP7B, the third ATP7B, the fourth ATP7B, the fifth ATP7B, the sixth ATP7B, and/or the seventh ATP7B signature peptides are lower than corresponding predetermined threshold concentrations or when the first ATP7B, the second ATP7B, the third ATP7B, the fourth ATP7B, the fifth ATP7B, the sixth ATP7B, and/or the seventh ATP7B signature peptides are absent.

176. The method of claim 175, wherein

the CD3ε signature peptide of SCID is encoded by an amino acid sequence set forth in SEQ ID NO: 1;

the first WASp signature peptide of WAS is encoded by an amino acid sequence set forth in SEQ ID NO: 2;

the second WASp signature peptide of WAS is encoded by an amino acid sequence set forth in SEQ ID NO: 3;

the first BTK signature peptide of XLA is encoded by an amino acid sequence set forth in SEQ ID NO: 4; and/or

the second BTK signature peptide of XLA is encoded by an amino acid sequence set forth in SEQ ID NO: 5;

the first CTNS signature peptide of cystinosis is encoded by an amino acid sequence set forth in SEQ ID NO: 6;

the second CTNS signature peptide of cystinosis is encoded by an amino acid sequence set forth in SEQ ID NO: 7 or 8;

the third CTNS signature peptide of cystinosis is encoded by an amino acid sequence set forth in SEQ ID NO: 12;

the fourth CTNS signature peptide of cystinosis is encoded by an amino acid sequence set forth in SEQ ID NO: 13;

the first SHPK signature peptide of cystinosis is encoded by an amino acid sequence set forth in SEQ ID NO: 9;

the second SHPK signature peptide of cystinosis is encoded by an amino acid sequence set forth in SEQ ID NO: 14;

the third SHPK signature peptide of cystinosis is encoded by an amino acid sequence set forth in SEQ ID NO: 15;

the first ATP7B signature peptide of WD is encoded by an amino acid sequence set forth in SEQ ID NO: 10;

the second ATP7B signature peptide of WD is encoded by an amino acid sequence set forth in SEQ ID NO: 11 or 21;

the third ATP7B signature peptide of WD is encoded by an amino acid sequence set forth in SEQ ID NO: 16;

the fourth ATP7B signature peptide of WD is encoded by an amino acid sequence set forth in SEQ ID NO: 17;

the fifth ATP7B signature peptide of WD is encoded by an amino acid sequence set forth in SEQ ID NO: 18;

the sixth ATP7B signature peptide of WD is encoded by an amino acid sequence set forth in SEQ ID NO: 19; and/or

the seventh ATP7B signature peptide of WD is encoded by an amino acid sequence set forth in SEQ ID NO: 20.

177. The method of claim 175, wherein

the antibody or antigen-binding fragment thereof that binds to the CD3ε signature peptide includes: a heavy chain variable (VH) domain including CDR1 of SEQ ID NO: 22, CDR2 of SEQ ID NO: 23, and CDR3 of SEQ ID NO: 24, and a light chain variable (VL) domain including: CDR1 of SEQ ID NO: 25, CDR2 of SEQ ID NO: 26, and CDR3 of SEQ ID NO: 27;

the antibody or antigen-binding fragment thereof that binds to the first WASp signature peptide includes: a heavy chain variable (VH) domain including CDR1 of SEQ ID NO: 28, CDR2 of SEQ ID NO: 29, and CDR3 of SEQ ID NO: 30, and a light chain variable (VL) domain including: CDR1 of SEQ ID NO: 31, CDR2 of SEQ ID NO: 32, and CDR3 of SEQ ID NO: 33; and/or

the antibody or antigen-binding fragment thereof that binds to the first BTK signature peptide includes: a heavy chain variable (VH) domain including CDR1 of SEQ ID NO: 34, CDR2 of SEQ ID NO: 35, and CDR3 of sequence GDI, and a light chain variable (VL) domain including: CDR1 of SEQ ID NO: 36, CDR2 of SEQ ID NO: 37, and CDR3 of SEQ ID NO: 38;

the antibody or antigen-binding fragment thereof that binds to the first CTNS signature peptide of cystinosis includes: a heavy chain variable (VH) domain including CDR1 of SEQ ID NO: 39, CDR2 of SEQ ID NO: 40, and CDR3 of SEQ ID NO: 41, and a light chain variable (VL) domain including: CDR1 of SEQ ID NO: 42, CDR2 of SEQ ID NO: 43, and CDR3 of SEQ ID NO: 44;

the antibody or antigen-binding fragment thereof that binds to the second CTNS signature peptide includes: a heavy chain variable (VH) domain including CDR1 of SEQ ID NO: 45, CDR2 of SEQ ID NO: 46, and CDR3 of SEQ ID NO: 47, and a light chain variable (VL) domain including: CDR1 of SEQ ID NO: 48, CDR2 of SEQ ID NO: 49, and CDR3 of SEQ ID NO: 50;

the antibody or antigen-binding fragment thereof that binds to the first SHPK signature peptide includes: a heavy chain variable (VH) domain including CDR1 of SEQ ID NO: 51, CDR2 of SEQ ID NO: 52, and CDR3 of SEQ ID NO: 53, and a light chain variable (VL) domain including: CDR1 of SEQ ID NO: 54, CDR2 of SEQ ID NO: 55, and CDR3 of SEQ ID NO: 56; and/or

the antibody or antigen-binding fragment thereof that binds to the second ATP7B signature peptide includes: a heavy chain variable (VH) domain including CDR1 of SEQ ID NO: 57, CDR2 of SEQ ID NO: 58, and CDR3 of SEQ ID NO: 59, and a light chain variable (VL) domain including: CDR1 of SEQ ID NO: 60, CDR2 of SEQ ID NO: 61, and CDR3 of SEQ ID NO: 62.